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Agilent 2100 Bioanalyzer User`s Guide

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1. Installation Verification Available Tests System Verification x Software Apply Description Executions Status Select All EX PC_MM_sk 1 of Executed passed E Files and Environment Analysis Calc 1 of Executed passed Unselect All EK Electrophoresis DNA ACE Prokaryote mRNA p Analysis Calc 1 of Executed passed vf ACE DNA 500 ACE Prokaryote mRNA n Analysis Calc 1 of Executed passed NES gt lt ACE DNA 1000 CE Eukaryote total RN Analysis Calc 1 X Executed Failed vf ACE DNA 7500 ACE Eukaryote total RN Analysis Calc 2 X Executed all executions failed 1e y ACE DNA 12000 ACE Prokaryote total R 1 of Executed passed FXK Electrophoresis RNA gt CE Prokaryote total R Analysis Calc 1 of Executed passed Comparison KS Test Properties Method Name ACE Prokaryote total RNA nano Description Analysis Calculation Engine Test electrophoresis Prokaryote total RNA nano Test checking features of analyses used with the Prokaryote total RNA nano assay Approximate Time 30s System lectrophoresis Proteit lowCytometry Assay Hardware L o __ Verification Run Complete amp Mr Advanced as Advanced Operator Auto Export Auto Print For both the bioanalyzer hardware and softw
2. All Instruments Instrument Diagnostics DEO2000386 M4 Instrument 2 Name Instrument 1 Firmware C 01 03q Serial DEO2000386 Product ID G29384 Available Tests Apply Name Description Status Start b xX Electronics Test Tests instrument electronics x Fan Test Tests if instrument Fan is working Fa x Lid Sensor Test Tests if the lid sensors are working z x Temperature Test Checks if the temperature sensors and heater a gt Unselect All Xx Stepper Motor Test Tests if horizontal and vertical motors are workin 7 aa Xx Electrode Diode Test Tests conductivity of channels pin to pin Xx High Yoltage Stability Test Tests high voltage accuracy and stability iwi UU Anm wams Chaski af tha hinh iinlbana canbeallae E Test Properties ID 29 Name Electronics Test Description Tests instrument electronics Approximate Time 5s 5 Click Start Contents A 322 V Index 6 Follow the instructions given by the 2100 expert software For example exchange the cartridge or put a test chip in the receptacle of the bioanalyzer when requested by the software Instrument Diagnostics Name Instrument 1 Serial DE02000386 Firmware C 01 03q Product ID G29384 Available Tests Apply Name Description Status x Electron
3. Auto Export Auto Print GM Advanced as Advanced Operator ie Offline Demo Mode The number of the sample that is currently being measured is indicated on the Reading Sample 4 information bar Running The status bar at the bottom of the screen shows the measurement progress for the chip run and the COM port number used for data acquisition Contents A65V Index During the chip run you can do the following e View the chip data file in the Data context by clicking on the name of the Data File COM Port Method Selection Start Stop Run Stop Method C Demo DNA 7500 Custom xsy Data File Ci 2005 05 19 14 01 59 xad e Switch to any other context For example you can evaluate any chip data file in the Data context or compare samples in the Comparison context e f necessary abort the chip run by clicking on the Stop button All data that was collected up to the stop point will be saved After the chip run is completed you can e Switch to the Data context where you can view analyze and evaluate the results of your chip run see Displaying the Measurement Results Electrophoresis on page 72 and Analyzing and Evaluating the Results of an Electrophoretic Assay on page 81 e Stay in the nstrument context and start a new assay for example Contents A 66 V Index Stopping a Chip Run You can stop a chip run at any time for
4. 7000 S semp 3000 a f0 Comparison O Sample 11 a Z 1500 Sample 12 1000 ak wag 1000 CS Ladder Tabs a S w Method _ a Sub tabs ma 30 EE aas a oe e SS oe ee t t 2 3 4 5 6 7 8 9 10 11 12 size bp Cone ng ul Molarity nmol Observations Lower 4 251 5 Lower Marker po z 74 8 80 179 7 Export 113 1 92 25 7 Configure Columns P a n e 165 0 98 8 7 Ee Copy To Clipboard Ctrl C 215 2 56 18 0 261 TA a 43 Manually Set Lower Marker 7 yl 310 1 98 3 7 al Manually Set Upper Marker 417 2 94 10 7 OF Exclude Peak cia Exes Er esults Peak Table Errors amp mr Advanced as Advanced Operator Setpoint Explorer Status Bar to Export Auto Print Contents A32V Index The 2100 expert software can be operated in six modes called contexts Instrument Context Data Context Verification Context Comparison Context Assay Context System Context NOTE The contexts work independent from each other regarding their data This means for example that you can review data and run measurements at the same time Contents A33 V Index Using the Contexts bar the Context menu or the selection list in the too bar you can switch between the contexts
5. Printer Help 4 You generally have the following possibilities Contents select the appropriate options select the items to be included in the report from the Print Item section select the wells to be included from the Wells section specify whether you want to generate the report as a file PDF or HTML A 288 V Index NOTE Your selections here are separate from the Auto Print selections they do not affect each other Both are used by default the next time you print even after restarting the program 5 Use the Page Setup and Printer buttons to access system dialog boxes allowing you to select a printer and specify the print medium and page layout 6 Click the Preview button to get a preview of the printouts or files to be generated 7 Click Print to print out the pages or generate the file s Contents A 289 V Index 2100 expert_mRNA Pico_00000_2005 05 17_15 59 32 xad 1 Assay Class mRNA Pico Page 1 of 16 Read 17 05 2005 15 59 41 Data Path Ci NA Pico 2100 expert_mRNA Pico_00000_2005 05 17_15 59 32 xad Modified 17 05 2005 16 02 27 Electrophoresis File Run Summary i Ladder Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 S a a jF a Instrument Information Sample 11 Assay Origin Path Instrument Name NJA NA Type UNKNOWN Firmware Simulation Mode Ci ProgrammetAgilenti2100 bioanalyzer 2100 expert Se
6. a Cells Apoptosis Apoptosis Demo Custom C a Cells 4poptosis Apoptosis Demo ers File Prefix 2100 expert max 25 charact Data Acquisition Parameters Run sample Bi to E E Default Fixed time 240 seconds max 300 sec Number of Cells 1000 events and no longer than 240 seconds max 300 sec 7 Ifrequired change the Data Acquisition Parameters a Enter the number of samples you want to be measured When preparing the chip see Preparing Samples and Chips for Flow Cytometric Assays on page 185 keep in mind that you have to follow the sequence of the sample wells For example if you want to measure 3 samples you have to fill the wells 1 2 and 3 with your samples and the remaining wells with cell buffer solution Contents A 183 V Index b Select the Data Acquisition Mode Select Default if you want the measurement time to be set to the default value 240 s sample The maximum time is shown in brackets 0R Select Fixed time and enter the time in s that the measurement of each sample is to take 0R Select Number of Cells and enter the minimum number of events that should be measured In the and no longer than field enter the maximum time in s a measurement can take regardless of whether or not the defined number of events is reached The maximum time is shown in brackets NOTE The overall run time for a chip is limited to 1440 s The individual run time f
7. i 5 Fluorescence D a value 102 1071 100 101 102 108 Fluorescence Contents A219 V Index How to Insert a Marker in a Histogram A marker is shown as two vertical lines that define a region of fluorescence values It is used to select a subset of events according to this fluorescence region NOTE You can insert markers only in generic assays To add a new marker 1 In the toolbar of the Histogram tab click the Insert Marker button A marker is added to the selected histogram window To insert an existing marker 1 Click the nsert Existing Marker button lil to open the nsert Existing Markers window Insert Existing Markers x Existing Markers Marker 2 2 Select a marker in the list and click Insert Marker The marker lines are added at the defined positions The label identifies the marker Contents A 220 V Index You can remove markers that you do not need any more 1 Click one of the vertical lines in the histogram to select the marker The lines of the selected marker are displayed bold No of Events gt D on o on on t N an Marker rrr trar o TIP You can also click the corresponding row in the result table to select the marker 2 Click the Delete Marker button mas to remove the marker Contents A221 V7 Index How to Configure Markers You can change the color name and the upper and lower limits of the marker 1 Do
8. m jadije eee T Agilent 2100 Bioanalyzer 2100 Expert User s Guide mom 000 Agilent Technologies Notices Agilent Technologies Inc 2000 2005 No part of this manual may be reproduced in any form or by any means including electronic storage and retrieval or translation into a foreign language without prior agreement and written consent from Agilent Technologies Inc as governed by United States and international copyright laws Manual Part number G2946 90004 Edition May 2005 Agilent Technologies Hewlett Packard Str 8 76337 Waldbronn Germany Adobe and Acrobat are U S registered trademarks of Adobe Systems Incorporated Microsoft and Windows are U S registered trademarks of Microsoft Corporation Technology Licenses The hardware and or software described in this document are furnished under a license and may be used or copied only in accordance with the terms of such license Warranty The material contained in this document is provided as is and is subject to being changed without notice in future editions Further to the maximum extent permitted by appli cable law Agilent disclaims all warranties either express or Contents A2V implied with regard to this manual and any information con tained herein including but not limited to the implied war ranties of merchantability and fitness for a particular purpose Agilent shall not be liable for errors or for inciden
9. Contexts E y Instrument Comparison X gt Method System NOTE Instrument F Comparison Method System Context View Gel v qa Verification he Data Verification Ie Comparison Method System Menus toolbars the tree view and the main working area tabs significantly change when you switch between the contexts An introduction to the six contexts is given in the following Contents A34V Index Instrument Context On startup 2100 expert enters the nstrument context where you can run DNA RNA protein or cell assays by selecting an assay file and starting the chip run provided that the bioanalyzer is properly connected a chip is inserted and the bioanalyzer lid is closed 42100 expert ETE File Context View Method Instrument Windows Help Instrument e i amp oa Contexts Demo DNA 7500 a Instruments Instrument Diagnostics Demo ene Instrument 2 iTS sealei Instrument 3 s Serial ean fa Method Selection be Methods amp Cartridge Data eia Start Stop Run Q start EZ Product ID Method C Demo DNA 7500 Custom xsy Verification Firmware Simulation Mode Data File 1e lt z A Assay Comments omparison a neat Copyright 2003 Agilent Technologies Default C sDNA DNA 7500 Demo DNA 7500 Custom Method Custom C sDNA DNA 7500Demo DNA 7500 Custom
10. If you switch to the Dot Plot tab one region is displayed in the dot plot The red fluorescence values of the region are related to the marker in the red histogram the blue fluorescence values to the marker in the blue histogram Corresponding to the histogram evaluation high blue fluorescence and high red fluorescence indicate living GFP producing cells See the following example Cumulation of high blue and high red fluorescence indicates living GFP expressing cells Sample 4 Region 1 Red Fluorescence a oO 107 107 107 10 10 10 10 10 10 Blue Fluorescence Contents A 256 V Index The results of the dot plot evaluation are displayed in the result table Events covered by the region All measured events All Events iepen Sevene E tote E of gated Amount of cells with high CBNF fluorescence and high GFP fluorescence in relation to all measured events Amount of cells with high GFP fluorescence in relation to the amount of CBNF stained cells Contents A 257 V Index Working with Chip Data and Assays You can make efficient use of the chip and assay data generated by the 2100 expert software if you know the following fundamentals and operating techniques e 2100 Expert Data Overview on page 259 e Handling Assays on page 262 e Handling Chip Data on page 267 e Organizing Backing up and Archiving 2100 Expert Data on page 269
11. No of Events 13 10 10 10 107 108 10 Fluorescence Contents A 210V Index Analyzing and Evaluating the Results of a Flow Cytometric Assay You can analyze and evaluate result data of flow cytometric assays using either the dot plot or the histogram view In both views you can evaluate the detected cells by defining areas of interest e Histograms show the distribution of events related to the red and blue fluorescence intensity Gating is used to generate subsets based on markers in one histogram See Using Histograms for Evaluation on page 212 for detailed information Dot plots show events as dots in a coordinate system where the blue fluorescence value is related to the red Regions and gates are used to determine the number of cells with a fluorescence intensity lying in a defined range See Using Dot Plots for Evaluation on page 233 for detailed information If you use predefined assays the markers and regions are set at the approximate position where the events are expected Refer to Evaluating Antibody Staining Apoptosis and GFP Assays on page 242 for information on how to evaluate the predefined assays TIP You can analyze and evaluate results while a chip run is still in progress Contents A211 V7 Index Using Histograms for Evaluation Histograms are graphical representations of the measurement results where the number of events cells is mapped to the Y axis and their fluorescenc
12. See Loading the Electrophoresis Chip into the Bioanalyzer on page 58 5 Start the chip run See Running an Electrophoretic Assay on page 64 When the chip run has finished you can e Have a first look at the results see Displaying the Measurement Results Electrophoresis on page 72 e Document the chip run see Entering Chip Sample and Study Information on page 69 e Analyze and evaluate the results See Analyzing and Evaluating the Results of an Electrophoretic Assay on page 81 See Result Flagging on page 152 Contents A50 V Index Selecting an Electrophoretic Assay for a Chip Run To select an assay 1 Switch to the nstrument context 2 Inthe Tree View Panel select the bioanalyzer you want to use g All Instruments y Instrument 1 v In the upper left of the nstrument tab an icon shows the status of the bioanalyzer You should see one of the following icons lid open closed indicating that the bioanalyzer is detected by the system 3 If you do not see one of these icons check that the bioanalyzer is switched on and properly connected Check the COM port setting Make sure the bioanalyzer is physically connected to the PC over the serial interface Check the power connection Check the power switch Contents Ab51lV Index cl If you need additional help please refer to the Agilent 2100 Bioanalyzer Maintenance and Troubleshooting Guide Sel
13. a fluoresces in blue and a H f red The height of the Intensity Intensity bars is related to the Histogram View number of cells with this fluorescence value Contents A 172 V Index In the histograms the bar chart is replaced by a point to point line as shown in the following image 5 24h fix Calcein Annexin CY5 250 200 a falcein SS SSS SS No of Events PA 8 i o gt iif o z a 4 eris d 0 T uu it ll J tit L LAL 102 104 100 10 10 10 Fluorescence Fluorescence For detailed information see Using Histograms for Evaluation on page 212 Contents A173 V Index Generating Dot Plots Single events can also be displayed related to both fluorescence values generating a map of dot plots In dot plot view the events cells with a minimum fluorescence intensity are displayed in a coordinate system logarithmic axis scaling Each axis represents a fluorescence color A high number of events cells with similar fluorescence values are displayed as a dense cluster of dots as shown in the following image As a cell passes through the detector its blue and red fluorescence values are measured Red Intensity Blue Intensity Dot Plot View To determine the number of cells whose blue and red fluorescence lies within a defined range you can insert regions Additionally a gate can be set for either red or blue flu
14. 1 From the Histogram or Dot Plot menu select Single View or Grid View 0R Click the Single View or Grid View button on the histogram dot plot toolbar OR Click the All Samples entry in the Tree View Panel to switch to the grid view or any sample to switch to the single view OR Double click any histogram or dot plot in the grid view to switch to single view The following example shows switching between grid view and single view for histograms o 104010 010 0 0 Fluorescence A 10 040 10 10 1010 Fluorescence Total events 1702 of gated 46 Total events 1752 of gated 45 3 4 24h fix 5 24h fix Calcein Annexin CYS Calcein Annexin t CYS Hf 2 v g 150 T i i S xini po 5 A 5 so Soe i t Z o4 F o 10510 0 40 1051010 1040 10 104040 10 Fluorescence Fluorescence luor Total events 1719 of gated 43 3 Total events 1736 of gated 44 5 Contents o 8 8 8 8 Histogram Calcein 8 rH E Palcein No of Events Fluorescence 2 24h fix Annexin CY5 Calcein 2 24h fix yd ee 10 f 10 10 Fluorescence Marker mn Max Events Marker Mn Max rents gt Al Events oo 7318 1702 p AlEvents 0 08 742 66 1702 Calcein on all events 6 88 298 01 93
15. File Prefix z100 expert max 16 characters System Data Acquisition Parameters Start Run Checklist E Is the instrument ready Run sample i fi Z v7 E hes of Is a chip detected of Is selected instrument valid For this assay of Does the cartridge and the selected assay match of Are all required licenses applied of 1s current instrument valid For the selected method C Does the user have rights to start a run Offline Demo Mode GM Advanced as Advanced Operator Auto Export Auto Print Contents A35V Index NOTE If two bioanalyzers are connected to your PC you can run both in parallel During the chip run s you can view the status of the bioanalyzer s instrument information and real time acquisition data In the nstrument context it is also possible to run hardware diagnostic tests on all connected bioanalyzers Refer to Running Instrument Diagnostics on page 316 for details Contents A 36 V Index Data Context In the Data context you can e view analyze and evaluate the results of your chip runs that are presented as electropherograms gel like images histograms dot plots and result tables e export and print the results of your chip runs 322100 expert C ea Data dsDNA DNA 7500 Demo DNA 7500 Custom 2100 expert_DNA 7500_00000_2005 05 19_14 01 59 xad File Context View Electropherogram Workflows Windows Help eH 92 kE S 2bJe 000 00 D8 PBa s NMA Bes amp lt gt CR Mix 3a N
16. The corresponding gating button in the toolbar is now enabled Click or to set the gating direction 0R Right click the marker in the histogram or in the result table and select Gate in Red Blue histogram from the context menu The gating direction is displayed in the Information Bar If the gating direction is already set you first have to remove the existing gating To remove gating 1 Click the Remove Gate button a The gating is removed and the corresponding gating button is enabled NOTE To change the gating direction in non generic assays you first have to change the assay to generic To achieve this use the mport Setpoints button on the Assay Properties tab refer to Importing Data on page 271 Contents A 226 V Index How to Overlay Histograms You can compare samples by overlaying their gated histograms This is useful for example if you want to see the progress of a reaction or if one sample is used as reference Overlaying histograms might also be helpful for adjusting the marker position You can overlay all measured samples Both red and blue histograms are overlaid NOTE You can configure the colors of the overlaid histograms and adjust the scale graduation as described in How to Set Signal Colors for Overlaid Histograms on page 229 To overlay histograms 1 2 3 Select the main sample and display the Histogram tab Click the Overlaid Samples 424 button to op
17. Using Log Books 2100 expert provides several log books to document all relevant actions and changes Due to requirements of data integrity and data security none of the log books can be cleared Run Logs The run log books can be found in the following contexts as sub tabs of the Log Book tab e Data context e Verification context e Comparison context e Assay context They contain events such as the start and end time of a chip run and any errors or problems that occurred during the run General Properties Assay Properties Chip Summary Gel Electropherogram Result Flagging l Log Book Time Stamp gt Demo Run Started File C Programme Agilent 2100 bioanalyzer 2100 Apr 27 2005 09 34 57 expert Secured4rea D ata dsDNA DNA 7500 Demo DNA 750052100 4 Demo Run ended Number of wells acquired 13 Instrument Run Apr 27 2005 09 36 49 All run logs are saved in the data files within the respective context Contents A310 V Index System Log The system log book can be found in the System context as a sub tab of the Log Book tab It includes start up and shut down events of the 2100 expert software and for example errors or problems with the connected bioanalyzers Users and Roles System Wide Settings Archiving Import Export System Log Book Time Stamp al System Log Signature Log Audit Trail Contents A 311V Description Number
18. Zero Baseline Contents A 361 V Index To remove the zeroing disable the Zero Baseline box in the setpoint explorer baseline calculation under Global and Advanced setting Fu IgG reduced 120 110 100 90 30 IgG reduced 70 60 50 40 30 None Zero Baseline Contents A 362 V Index Index A Accessories 334 Adding regions 234 Agilent Online Store 334 Alignment 128 Antibody staining 243 APC 169 Apoptosis assays 247 Assay setpoints 262 Assays Creating new 264 Generic 178 Opening 265 Predefined 176 Stopping 67 200 Auto Print 304 Auto Run 304 B Base pair 352 Baseline 124 Bioanalyzer manuals 8 Bioanalyzer tests 317 Bubbles how to avoid 187 Contents c Calcein 168 Capillary electrophoresis 352 Cartridges 58 189 CBNF 169 Cell detection 170 Chip reagents 186 Color overlaid histograms 229 Comparing samples 143 Comparison context 143 Configuring markers 222 Context definition 33 Copy Markers 225 Regions 239 Creating new assays 264 Cy5 169 D Data specify file names and directories 300 Data analysis setpoints 116 A 363 V Data analysis setpoints 262 Data files 260 Data points 76 208 Documentation related 8 Dot Plot generating 174 Dyes 168 E Electrode cartridge 58 Electrodes 60 190 Electrokinetic forces 346 Electroosmotic Flow 346 Electrophoresis 346 Electrophoretic flow 346 Event 171 F Fluor
19. e zoom enlarge or reduce using the mouse the graphs to display details for example In electropherograms you can additionally e show data points e pan and scale the graph using the mouse e change the background from a gray to white gradient to white e add a grid to the electropherograms In gel like images you can additionally e change the gel color e change order of gel lanes in gel like images Contents AlV Index To zoom into an electropherogram 1 From the Electropherogram menu select Graph Mode gt Zoom default setting 2 Position the mouse pointer in the electropherogram 3 Click and hold down the left mouse button The mouse pointer changes its shape to a magnifying glass Qo 4 Drag the mouse A rectangle shows the part of the an electropherogram to be enlarged 5 Release the mouse button FU FU oot sot 80 707 607 f PCR Mix 2 N w D a a a PCR Mix 2 en i ett ns anette at S a i 40 50 60 70 80 90 100 Contents A116 V Index To pan and scale an electropherogram 1 From the Electropherogram menu select Graph Mode gt Pan or Scale 2 Position the mouse pointer in the electropherogram 3 Click and hold down the left mouse button The mouse pointer changes its shape to a double arrow or to a double crosshair 4 Drag the mouse As you drag the mouse the electropherogram curve moves in the dra
20. tal or consequential damages in connection with the fur nishing use or performance of this document or of any information contained herein Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms the warranty terms in the separate agreement shall control Restricted Rights Legend If software is for use in the performance of a U S Govern ment prime contract or subcontract Software is delivered and licensed as Commercial computer software as defined in DFAR 252 227 7014 June 1995 or as a com mercial item as defined in FAR 2 101 a or as Restricted computer software as defined in FAR 52 227 19 June 1987 or any equivalent agency regulation or contract clause Use duplication or disclosure of Software is subject to Agilent Technologies standard commercial license terms and non DOD Departments and Agencies of the U S Government will receive no greater than Restricted Rights as defined in FAR 52 227 19 c 1 2 June 1987 U S Gov ernment users will receive no greater than Limited Rights as defined in FAR 52 227 14 June 1987 or DFAR 252 227 7015 b 2 November 1995 as applicable in any technical data Index Contents Abo t this Man al sccictsscscitviewdciecticiunscnicnnistierevieteinnnaneiole neice eeeiedentiee 5 Mths Mangal at cae E cae te pec aca eee ee 6 Related DOCUMENTS vscssaxtecsacescectccaensaxsc
21. 107 10 10 10 10 Blue Fluorescence Contents A 251V Index The results of the dot plot evaluation are displayed in the result table All measured events Events covered by the region Region Events total__ of gated _ ize 4 Jy Amount of living cells with high red fluorescence in relation to the amount of all cells Amount of living cells with high red fluorescence in relation to the amount of living cells Contents A252 V Index Evaluating GFP Assays With GFP Green Fluorescent Protein assays the fluorescent substance is not a dye but a protein Cells can be transfected with a target gene together with the GFP producing gene Transfected cells produce the fluorescent protein which can be detected The fluorescence shows you the success of the transfection experiment For detailed information on GFP assays refer to the application note Monitoring transfection efficiency by green fluorescent protein GFP detection with the Agilent 2100 Bioanalyzer For a detailed description on how to evaluate the results using histograms and regions refer to Using Histograms for Evaluation on page 212 and Using Dot Plots for Evaluation on page 233 Gating direction The GFP has a green fluorescence absorption in the blue Because the reference dye CBNF fluoresces in the red the gating direction is from red to blue CBNF stains living cells so you can detect living GFP positi
22. 45 0 OO 0 0 System Peak 18 4 7 4 43 2 Calibrated Protein 29 1 oo 568 Ie LD 1000 OO 0 0 Upper Marker Contents A110 V Index The calibration curve can be displayed by switching to the Calibration Curve sub tab on the Chip Summary tab 1200 1000 D co S Relative Conc pg ml 8 50 100 150 alibration Curve Y 2 99 X R 2 0 9978 200 250 300 Standard Conc ug ml Sample Information Instrument Information Standard Curve ii 350 400 Contents A111 V7 Index Smear Analysis The 2100 expert software allows to perform a smear analysis for all electrophoresis assays When the smear analysis is enabled the software allows you to define regions of interest These regions are used to define the area of broad peaks and determine their part of the total area Smear analysis provide a means to analyze broad signals that can be hardly evaluated with the normal peak assignment You therefore can define regions of interest that contain the peaks base pair size that you are interested in For these regions you can determine the covered area in relation to the total area Enabling Smear Analysis To enable smear analysis 1 Go to the Electropherogram tab in the Data context 2 Go to the setpoint explorer and select the Local or Global tab depending on which samples should be analyzed 3 Select the Advanced mode Contents A 112 V Index 4 Under Smear
23. Area Threshold The Area Threshold determines the minimum amount of peak area that must be detected before a peak is recognized Height Threshold The Height Threshold setpoint determines whether a peak is kept It represents the minimal peak height For each peak the difference between the start point value and the center point value local baseline must be greater than the Height Threshold value Peak Filter Width The Peak Filter Width setpoint determines the minimum amount of time that must elapse before a peak is recognized Baseline Plateau The Baseline Plateau setpoint is a parameter that assists in finding peaks The signal is recognized to be at baseline whenever the slope of the data is less than the Slope Threshold setpoint either positive or negative for longer than the time set for the Baseline Plateau This setting rejects brief low slope areas such as between non baseline resolved peaks Contents A122 V Index Manually Moving Fragment Start and End Points RNA and Cy5 Labeled Nucleic Acids It is also possible to alter the start and end points manually for individual fragments in an RNA or Cy5 labeled nucleic acids assay The integration borders of detected RNA fragments are displayed in the Fragment Table sub tab Zooming in on the base of a particular fragment allows you to see the start and end points Placing the cursor over one of these points changes the cursor to a pointing hand allowing you to click and drag the poi
24. NOTE If the AutoRun option is active the chip run starts automatically once a chip has been detected and the lid has been closed Contents A63 V Index Running an Electrophoretic Assay NOTE You can stop a chip run at any time for example if errors occurred or if you are not satisfied with the quality of the measurement results that you can observe during the chip run See Stopping a Chip Run on page 67 Starting the Chip Run When you have loaded the chip you can start the chip run 1 On the nstrument tab click the Start button start The chip run starts The Raw Signals sub tab shows an electropherogram of the currently measured sample The name of the sample is displayed above the graph The graph is a live plot of the migration time against fluorescence units raw data including background fluorescence for example Contents Ab4V Index 322100 expert File Context View Method Instrument Windows Help Instrument e E amp oa Demo DNA 7500 All Instruments i Ef Demo 2100 expert_DN Instrument 2 Diagnostics Instrument Instrument 3 oun j 3 Method Selection Methods ia 2 g Cartridge E P Start Stop Run Stop Data E Vendor KA DNA Labchip Product ID Method C Demo DNA 7500 Custom xsy E3 r zs Firmware Simulation Mode Data File Crh 2005 05 19 14 01 59 xad Verification 1e lt Comparison CS Method System Fluorescence
25. Sample Name Sample Comment Rest Digest Status Observation Result Label Result Color 1 PCRMix1 25 35 50 53 70 M v 2 PcRMix2 150 158 200 210 M v FoundLadder2 3 PCR Mix 3 500 550 600 650 Vv wy Found Ladder 3 4 PCRMix1 25 35 50 53 70 M v 5 PCRMix2 150 158 200 210 d v Foundladder2 6 PCRMx3 500 550 600 650 M v Found Ladder 3 7 _ PCRMix1 25 35 50 53 70 M v 8 PCRMx2 150 158 200 210 M v FoundLadder2 M 9 PcRmix3 500 550 600 650 M v Found Ladder 3 10 PCRMix1 25 35 50 53 70 M v pIPCRMix2 150 158 200 210 Mi v FoundLadder2 M 12 PCRMix3 500 550 600 650 M v Found Ladder 3 Chip Lot Reagent Kit Lot 223 29 Sample Information Instrument Information Standard Curve Import Export The colors in the Result Flagging column show which sample matches which rule Contents A 157 V Index e On the Gel Tab General Properties Assay Properties Chip Summar Electropherogram Result Flagging Log Book l bp Ladder PCR PCR PCR PCR PCR PCR PCR PCR PCR PCR PCR PCR bp a EE E E a 10380 m m ee p e e a a a ooon _ 7000 6 0 00 fe 3000 w __ os Reaur 0 0 0 a p p 1000 700 ee ee 7 500 0 wA ss Ban EE a ce Se
26. changed in size and position until they include a specific event subset As a result you get the number of cells included in the region related to the total number of cells NOTE You can add remove regions and gates only in Generic assays Additionally you can insert a horizontal or a vertical gate for one region This is useful for counting all cells that have fluorescence intensities within the horizontal or vertical borders of the region In predefined assays the vertical side of a region corresponds to the marker of the blue histogram the horizontal side to the red one see Using Histograms for Evaluation on page 212 The gate is always displayed and corresponds to the range of the marker that is used for gating If you move a marker in a histogram the region and gate are automatically updated If you change a region or gate the marker is also updated Statistics are displayed in the result table below the dot plot Contents A 233 V Index How to Add Regions to Dot Plots Generic Assay only You can draw regions in dot plots of generic assays If there are regions already defined in other samples you can copy these regions in the dot plot of the current sample To draw a new region 1 Click the Insert Region button E in the toolbar The mouse pointer changes its shape to a crosshair 2 Draw a rectangle into the dot plot New regions are automatically named Region x where x is an auto incremented number By default th
27. n a g N i Size kDa Rel Conc ug ml Calib Conc ug ml Total Observations Area 1 a 6 0 0 0 0 0 0 0 Lower Marker 19 9 2 8 5 0 0 0 0 0 0 System Peak 27 5 Sm 25 3 140 7 0 0 23 7 38 5 4 58 1 451 7 0 0 76 3 32 6 5 gt 210 0 100 0 0 0 0 0 Upper Marker 10 3 NOTE The software performs alignment by default Turning automatic data analysis off suspends analysis until you turn it on again Contents A 107 V Index 7 The standard curve in conjunction with the markers is used to calculate protein sizes for each sample from the migration times measured 8 To calculate the concentration of the individual proteins in all sample wells the upper marker is applied to the individual sample peaks in all sample wells NOTE The software allows you to define upper and lower markers yourself A change in the selection of the markers will lead to quantitative changes in the calibration procedure however and therefore in the entire data evaluation Contents A 108 V Index Protein Absolute Quantitation Absolute quantitation is calculated based on the relative concentration of a sample and the user defined standard and the known concentration of this user defined standard For protein samples you can enable the use of calibration for each sample and enter the concentration of the standard protein This allows you to generate a calibration curve which can be used to
28. 0 System Peak aes T O ste Approval Contents A 137V Index 3 Two baseline points and the connecting line will appear and the integration results shown in the result and peak tables will be updated Size kDa Rel Conc ug ml Calib Conc ug ml Total Observations 6 0 0 0 0 0 0 0 Lower Marker 0 0 0 0 System Peak 0 0 0 0 System Peak 224 9 100 0 Calibration Protein 0 0 Upper Marker Contents A 138 V Index TIP If you want to change several baseline points deactivate the automatic analysis by clicking the Pause Analysis button in the toolbar This way the software will not recalculate the data analysis with every change Once you have changed all baseline points click the Pause Analysis button again to activate automatic analysis 4 Adjust the baseline points as appropriate TIP To move a peak baseline point along the vertical line press the CTRL key and the left mouse button To move a peak baseline point along the signal press the left mouse button only Contents A 139 V Index 5 Click the Automatic Analysis button S to enable the integration again The integration results in the result and peak tables will change according to the changes done Sample 3 Size kDa Rel Conc ug ml Calib Conc ug ml Total Observations 14 6 0 0 0 0 0 0 0 Lower Marker 2 8 3 0 0 0 0 0 0 System Peak 3 3 5 0 0 0 0 0 0 System
29. 1 Go to your desktop and double click the following icon sie 2100 ma 0R From the Windows Start menu select Programs gt Agilent 2100 Bioanalyzer gt 2100 expert Contents A 31y Index 2100 Expert Work Area The 2100 expert software has standard elements such as pull down menus and toolbars and the main working area which contains several tabs some of which have sub tabs The 2100 expert work area has the following regions demonstrated at the Data context Title Bar 2100 expert C ea Data dsDNA DNA 7500 Demo DNA 7500 Custom 2100 expert_DNA 7500_00000_2005 05 19 14 01 59 xad Menu Bar Context View Gel Workflows Windows Help eH 9e kE ee Bue COO0 O0 SB BaF e 2 Toolbar Gel CR Mix 3a Not Reviewed Hig All Files General Properties Assay Properties Chip Summary Gel Electropherogram Result Flagging Log Book a B K 2100 expert_DNA 750p Info Bar ee All Samples bp tas P Pro S8 0 58 Sh Sa Sa Sa Sa Sa 58 5 Bp Local Global Mix 3a Normal bd Collapse A gt Sample 3 Integration start time s 26 Context Bar_ es Sample 4 Integration end time s 94 KA L OQ Sample 5 Slope Threshold 0 5 Odc dc d d Sample 6 Area threshold 05 Verification Ame Height threshold FU 8 T Vi Sample 8 10380 me a es ee 10380 Peak filter width s 0 5 re e l ew E Sample 9 7000
30. 98 V Index Troubleshooting the RIN To obtain meaninful and reproducible results the lower marker and ribosomal bands must be identified correctly Although the ribosomal fragment identification has been improved in rare cases i e when analyzing degraded RNA samples the fragment baseline is not properly set In this case the user should adjust the baseline settings manually Example Incorrect software identification of the ribosomal fragments RIN 7 2 Sample 7 Contents AID V Index RNA integrity number after the manual adjustment RIN 5 7 On details on how to adjust the lower marker and ribosomal bands please refer to Changing the Data Analysis on page 116 Contents A 100 V Index Use Models for the RNA Integrity Number To take full advantage of the RIN feature a 2 step use model is suggested 1 Determine the threshold value for the RIN that results in meaningful downstream experiments a Culture Isolation of total RNA RNA QC via oe Agilent 2100 bioanalyzer Correlate RIN with downstream experiment and determine threshold RIN for meaningful results iterative process Contents A101 V7 Index 2 Run standard experiment and use RIN to determine if sample integrity is sufficient Cells Culture j Isolation of total RNA RIN RNA OC via sided y Agilent 2100 bioanalyzer threshold 7 RIN above threshold Continue with downstream exp
31. All events in relation to the blue marker here calcein ate l tevens Zisa horami gt All Events Living cells in relation to all measured cells high calcein fluorescence When using the calcein marker in the blue histogram for gating only the living cells are considered for building the red histogram High red fluorescence values indicate living apoptotic cells low red fluorescence values indicate living non apoptotic cells See the following example Contents A249 V Index Annexin CY5 High fluorescence value indicates living apoptotic cells 2 o WwW mm 5 z Low fluorescence value indicates living non apoptotic cells Percentage of all cells with high red fluorescence selected by the red marker Amount of living cells with high red fluorescence in relation to the amount of living cells Contents A 250 V Index Dot plot evaluation If you switch to the Dot Plot tab one region is displayed in the dot plot The red fluorescence values of the region are related to the marker in the red histogram the blue fluorescence values to the marker in the blue histogram As in the histogram evaluation high blue fluorescence and high red fluorescence represent living cells with annexin V binding See the following example Cumulation of high blue and high red fluorescence indicate living apoptotic cells 5 24h fix Red Fluorescence LLL LLL Loio LLL miau 10
32. Analysis select the check box Perform Smear Analysis Local Global Advanced Align to lower marker Quantitation Concentration of upper Concentration of lower Sizing Standard Curve Smear Analysis Baseline calculation Baseline start time s Baseline end time s Zero Baseline Filter Settings Filter width s Polynomial order Baseline correction Perform Baseline Corre Integrator Integration start time s Integration end time s Slope Threshold Area threshold Height threshold FU Peak filter width s Baseline plateau s Width threshold s Peak filter polynom Ladder Setpoints Baseline calculation Baseline start time s Baseline end time s L X Collapse x al 4 2 8 3 Point to Point 26 94 x 0 5 4 m 26 94 0 5 0 5 8 0 5 5 1 6 26 94 xl Defauts Help The Region Table sub tab is added to the Electropherogram tab Contents A 113 V Index Performing Smear Analysis After enabling the smear analysis in the setpoint explorer you are able to insert regions of interest in the electropherogram To do so 1 2 Select the Region Table sub tab in the Electropherogram tab Right click the electropherogram and select Add region A region will be inserted into the electropherogram The Region Table shows the values for the inserted region Repeat the previous step until the number of required regions
33. Contents A192 V Index CAUTION Do not force the chip selector handle when a chip is inserted in the bioanalyzer 3 Place the prepared chip into the receptacle The chip fits only one way Do not use force Cell chip Chip selector in position 2 Contents A 193 V Index 4 Carefully close the lid CAUTION Do not force the lid closed This can damage the pressure cartridge If the lid does not close without force check that chip is inserted correctly and that the chip selector is at the correct position for this chip type When the software recognizes an inserted chip the chip is shown on the Instrument tab If you have closed the lid and the software has not recognized the chip verify that the cartridge and chip are inserted properly and the chip selector is in the correct position Close the lid The adapter with the gasket in the cartridge fits onto the priming well of the chip A small gap between the lid and the instrument mainframe is normal and no cause for malfunction The icon on the nstrument tab changes to a cell chip icon Cell LabChip If the chip is not detected open and close the lid again F 2 ee 3s bch Y sody e 2 P Contents A19 Index NOTE If the AutoRun option is active the chip run starts automatically once a chip has been inserted and the lid has been closed Contents A 195 V Index Running a Flow Cytometric Assay Running a flow cytometric
34. GIF images are limited to 256 colors 8 bit JPEG images may contain millions of colors 24 bit as well as additional information including PostScript clipping paths Contents A 349 V Index L Lab on a chip The generic term for a microfluidic product signifying a chemical process or material movement taking place on a microchip In contrast to analysis in a standard laboratory that relies on human intervention at several stages to manipulate or observe samples and record results the self contained lab on a chip represents an almost hands free technology Lab on a chip technology means downsizing of analytical techniques from lab scale to chip scale e using techniques like electrophoresis chromatography and sieving e with fluorescence absorbance and MS detection e with a higher degree of automation integrating multiple steps of a complex protocol into a miniaturized system Virtually any biochemical testing that can be done in a laboratory can theoretically be done on a chip Ladder Each electrophoretic LabChip Reagent kit contains a ladder A ladder contains DNA RNA fragments or proteins of known sizes and concentrations A ladder well is located at the bottom right of the chip The ladder is analyzed first before sample analysis takes place Contents A 350 V Index The peak sizes and markers defined for the ladder are assigned consecutively starting with the first peak detected in the ladder Peaks appearing abo
35. Mix 1 25 35 50 53 70 m m Not Reviewed 11 PCR Mix 2 150 158 200 210 m ww A Not Reviewed P PCR Mix 3 500 550 600 650 m m A Not Reviewed Chip Lot Reagent Kit Lot 223 29 Chip Comments prepared by laural I Sample Information l Instrument Information Standard Curve Import Export NO TE You may find some input fields already filled in because chip sample and study information are taken over from the base assay or chip data file Contents Al0V Index 3 From the File menu select Save TIP You can import chip sample and study information from txt or csv files This is especially helpful and time saving if you already have documented a similar chip run in another chip data file Refer to Importing Chip Sample and Study Information on page 275 for details Contents Any Index Displaying the Measurement Results Electrophoresis You can view the measurement results of an electrophoretic chip run as electropherograms or gel like images e You can display the electropherograms either one sample at a time or all samples at the same time to get an overview of the chip run for example to see the progress of a reaction See How to Switch Between Single View and Grid View Electropherograms on page 73 You can navigate through the samples See How to Navigate Through the Samples on page 74 You can change the display of electropherograms and gel
36. Peak 100 0 Calibration Protein 0 0 Upper Marker Contents A 140 V Index Reanalyzing a Chip Data File NOTE Occasionally you may wish to open and view or reanalyze a chip data file that was run and saved previously The raw data values are saved in the data file along with the analysis settings that were chosen for the run so that the data can be reanalyzed with different settings To do this 1 Click File gt Open to open a chip data file xad 2 Choose the filename from the list of data files 3 Click OK The items that can be changed for reanalysis are All Electrophoresis Assays e Global peak find settings e Individual sample peak find settings e Gel color e Sample names and comments RNA Assays Only e Fragment names and colors associated with labels e Fragment start end times additional fragments or delete fragments found Contents A 141V Index e Reassign lower marker DNA and Protein Assays Only e Exclude peaks from analysis e Reassign upper lower markers e Alignment or no alignment with marker peaks e Manual integration Protein Assays Only e Absolute quantitation TIP When applying modified data analysis setpoints the software will by default immediately recalculate the raw data which takes some time Select Don t Analyze from the Gel Menu or Electropherogram Menu to temporarily switch off automatic data analysis while you modify setpoints If you save the data file afte
37. Result Color Approved LI Sample 12 PCRMix1 ref wv A Not Reviewed Ladder 3a m m Not Reviewed Sample 3 m m A Not Reviewed Method Sample 4 m M Not Reviewed Sample 5 m vy Not Reviewed Sample 6 m y A Not Reviewed System Sample 7 Oo ww A Not Reviewed Sample 8 m y A Not Reviewed Sample 9 m y A Not Reviewed Sample 10 N O vy A Not Reviewed Sample 11 m Ay A Not Reviewed Sample 12 wv A Not Reviewed Chip Lot Reagent Kit Lot Chip Comments prepared by laura Sample Information l Instrument Information Standard Curve Import Export Mr Advanced as Advanced Operator Auto Export Auto Print Contents A2V Index 7 In the tree view panel click any sample name or the ladder This selects the Electropherogram tab which displays a data plot of size migration time versus fluorescence intensity 42100 expert C ea Data dsDNA DNA 7500 Demo DNA 7500 Custom 2100 expert_DNA 7500_00000_ 2005 05 19 14 01 59 xad File Context View Electropherogram Workflows Windows Help Data eu 9e EE S ua C00 CO n0al AeH S ANM eR contexts Ele CR Mix 3a Not Reviewed e All Files General Properties l Assay Properties Chip Summary Gel Electropherogram Result Flagging Log Book E Hg 2100 expert_DNA 7500_001 Instrument E FU PCR Mix 3a mj fim oe E V O Sample 3 Data mj Sample
38. Right click the graph or table region 2 From the context menu select Copy Gel Copy Electropherogram graphs or Copy To Clipboard tables 0R Click the button in the toolbar Contents A 283 V Index You can now switch to a word processing spreadsheet graphics or other application and paste the graph or table there Exporting Chip Sample and Study Information On the Sample Information and Study Information sub tabs of the Chip Summary tab you can enter names and comments regarding chip samples and study The information you enter here may be very similar for further chip runs or other assays Once you have entered the information you can export it into a separate file which you can then import into other chip data xad or assay xsy files instead of typing it anew The import export files can have the extension txt or csv and have a fixed form which differs for electrophoretic and flow cytometric assays To export chip sample and study information to a file 1 On the Chip Summary tab click Export The Export Sample Information dialog box appears 2 Specify a file name and location for the file to which you want to export 3 Click Save Contents A284 V Index Exporting Result Flagging Rules You can export result flagging rules for reuse in other electrophoretic assay xsy or chip data xad files see Importing Result Flagging Rules on page 2 6 Result flagging rules are stored in x
39. SS l T M L 1 2 3 4 5 6 7 8 9 10 11 12 The spot on top of the lane is colored if the sample matches a result flagging rule e On the small gel image on the Lower Panel e088 O88 Li2 456 7 8 9 101112 Contents A 158 V Index e On the Results tab Number of peaks found 10 Result Flagging Color Result Flagging Label Found Ladder 3 Result Flagging Color color of the result flagging rule that the current sample matches Result Flagging Label label of the result flagging rule that the current sample matches How to Use the Form Mode The Form Mode provides some pre defined rules forms that you can use to define the result flagging rules to color code your samples You can set up any number of rules for evaluation As a typical example of how these forms are used you can use the form mode to flag DNA samples that have a fragment purity of 10 for fragment sizes of 150 bp To do this proceed as follows 1 Open the job that contains the results to be analyzed in the Data context and switch to the Result Flagging tab 2 Switch to the form mode by clicking the Switch to Form Mode icon E3 3 Choose the Search Fragment with Purity form from the Select Form selection list Contents A 159 V Index The Search Fragment with Purity form is displayed General Properties Assay Properties Chip Summary Gel Electropherogram Result Flagging Log Book Search for one or more specific fragmen
40. Source Category Sub Category amp File has been modified C Programme Agilent 2100 bioanalyzer2100 3 Security Service System Apr 26 2005 08 10 42 7 expert Secured amp reaSystem SystemFile xml amp File has been modified C Programme Agilent 2100 bioanalyzer 2100 3 Security Service System Apr 26 2005 08 10 40 __ expert Secured amp reaSystem SystemFile xml User switched new user is PC_MM_SK advaoper and old user was PC_LMM_SK 2100admin 0 User Interface Apr 26 2005 08 10 20 File has been modified C Programme Agilent2100 bioanalyzer 2100 3 Security Service System Apr 26 2005 08 09 12 expert Secured4reaMethod T emplates RNASEukaryote Total RNA Nano xsy File has been modified C Programme Agilent 21 00 bioanalyzer 2100 3 Security Service System Apr 26 2005 08 09 12 7 _expet Secured rea rchive File has been modified C Programme Agilent 2100 bioanalyzer 2100 3 Security Service System Apr 26 2005 08 09 11 expert S ecured4readrchive File has been modified C Programme Agilent 2100 bioanalyzer 2100 3 Security Service System Apr 26 2005 08 09 10 expert S ecured4readrchive File has been modified C Programme 4gilent2100 bioanalyzer 2100 3 Security Service System Apr26 2005 08 09 07 expert Secured4reaS ystem SystemFile xml File has been modified C Programme Agilent 21 00 bioanalyzer 2100 3 Security Service System Apr26 2005 08 09 06 expert Secured4reaS ystem SystemFile xml File has been modified C Programme Ag
41. Tab is enabled the 2100 expert software flags peaks that may have co migrated Size bp Conc najul Molarity nmol l Observations 1 4 15 4 20 424 2 Lower Marker 2 22 1 71 116 6 Possible Co Migration of 4 Peaks a 55 1 31 35 8 4 104 4 22 61 3 Possible Co Migration of 2 Peaks 5 141 3 19 34 4 bel s 38 315 z 235 4 74 30 6 ja 330 6 81 31 3 9 381 7 99 31 8 10 476 10 34 32 9 11 512 9 31 27 6 12 gt 1 500 2 10 2 1 Upper Marker Results Peak Table Legend Errors Since it is assumed that the molarity of all the fragments in a restriction digest should be the same any peaks or clusters having a molarity that is significantly larger than the rest are flagged as potentially co migrating peaks allowing you to examine them in more detail Contents A 86 V Index Data Analysis RNA and Cy5 Labeled Nucleic Acids The data analysis process for RNA and the Cy5 labeled nucleic acids assays consists of the following steps 1 Raw data is read and stored by the system for all of the individual samples 2 The data is filtered and the resulting electropherograms of all samples are plotted You can change the settings of the data analysis after the run and reanalyze your data 3 Fragments are identified for all samples and tabulated by peak ID You can change the settings of the peak find algorithm for any or all samples and reanalyze the data 4 An
42. Test Lid Sensor Test Stepper Motor Test Temperature Test Verifies proper functioning of all electronic boards in the bioanalyzer Checks if the fan is running at the appropriate speed Verifies proper operation of the lid sensor ensuring that the laser and LED are off when the lid is open Checks for proper movement of the stepper motor Checks if the temperature ramp up speed of the heater plate is within specifications Contents A 317 V Index Electrode Cartridge Tests Diagnostics Test Purpose HV Stability and Accuracy Test HV Accuracy Test On Load Short Circuit Test Electrode Diode Test Optics Test Electrophoresis Autofocus Test Laser Stability Test Tests high voltage accuracy and stability of all 16 high voltage power supplies and the high voltage controller Unused chip DNA RNA or protein required Check of channel reference diode in transmission direction Checks for instrument leak currents using an empty chip Note the limits of this test specify an ambient temperature of 25 C and relative humidity less than or equal to 60 Higher temperatures or relative humidity could result in a leak current Checks the photo diode and current versus voltage performance of the bioanalyzer Electrode diode test chip required Checks for proper alignment of internal optics and proper function of the laser and LED Checks the focusing capability of the optical system Au
43. To Clipboard Ctrl C att Scale to Selected Peak 4g Manually Set Lower Marker Tang Exclude Peal CAUTION Excluding a peak or manually setting a peak to be an upper or lower marker may cause errors during analysis Contents A121 V Index Aligning or Unaligning the Marker Peaks The upper and lower are then aligned to the ladder markers by resampling the sample data in a linear stretch or compression using a point to point fit Data before alignment Pro Pro Ig Pro La Contents A128 V Index Markers aligned to the ladder If the sample marker peaks are either more than twice as far apart or less than half as far apart as the ladder markers they are assumed to be the wrong peaks and analysis of the sample stops producing the error Marker peaks not detected Contents A129 V Index NOTE With DNA and protein assays the height of marker peaks is assay dependent Ladder peaks are analyzed to calculate a marker peak threshold that is used to locate the marker peaks in the sample wells If the marker peaks found using this calculated method fail to align with those of a sample the 2100 expert software will use the minimum peak height threshold setting instead if this value is lower than the value for the marker peak For example the calculated threshold might be too high to find the sample s markers if they happen to be very small for some reason Either no markers will be found or the wrong p
44. V Index How to Set Run and Result Options You can select several options such as to pause the analysis on setpoint changes the maximum log file size or the graph colors To set the Run and Result options 1 Switch to the System context and select the System Wide Settings tab 2 Select Run and Result in the tree navigation The Run and Result screen becomes visible Advanced J7 Limit the storage of system log Upper Limit in MB 5 Clear Log T Auto Run JT Auto Print Settings Analysis J7 Pause Analysis on Setpoint Change Graph Signal Color Signal M Sinal5 i Sigal Signal2 M Signal 6 M Signal 10 Signal3 Sina Signal 11 Signal 4 Signal 8 M Signal 12 7 3 Inthe Advanced section you can select Limit the storage of system log if you want to limit the disk space for the system log file SystemLogBook log located in the log subdirectory and enter an upper limit in MB Contents A 303 V Index If the limit is exceeded a message appears that prompts you to delete or move the log file to get free disk space select Auto Run to activate the automatic start of a chip run once the lid of the Agilent 2100 bioanalyzer is closed and a chip suiting the selected assay is detected select Auto Print to enable the automatic report printing function You can now click Settings to display the Autoprint dialog box where you set the options for automatic printing after a chip ru
45. analyze and quantitate this protein within different samples on the same chip Using Calibration in Protein Assays The calibration feature for protein assays allows absolute quantitation based on external standard calibration On the Chip Summary tab use the sample table on the Sample Information sub tab to define the samples that you want to use as calibration standards and enter a concentration Sample Name Sample Comment Use For Calibration Conc ug ml Status 1 betaLe m o wv 2 betaLG M 70a 3 beta LG M 140 l gt beta LG M y 5 ovalbumin NR non reduced O Ol y 6 ovalbumin R reduced i Jaj Ol y 7 insulin B chain 20 ug ml O Ow 8 standard O 0 y 9 betaLG 500 ug ml Vv 120 vy 10 standard B O Ol wy Contents A 109 V Index The calibration standard should be run in different concentrations to generate a calibration curve The software will automatically produce this calibration curve based on these inputs to determine the actual concentration of samples within the same chip In the peak tables of the samples a remark is added to the observation column to identify the calibration protein and the calibrated proteins Total Observations 0 0 Lower Marker 0 0 0 0 System Peak System Peak 0 0 J 00 e a 83 7 Calibration Protein 96 R 8 0 Upper Marker Observations 4 0 0 0 0 0 0 Po el 0 0 0 0 0 0 System Peak 3
46. area under the ladder is compared with the sum of the sample peak areas The area under the lower marker is not taken into consideration For total RNA assays the ribosomal ratio is determined giving an indication on the integrity of the RNA sample Additionally the RNA integrity number RIN can be utilized to estimate the integrity of total RNA samples based on the entire electrophoretic trace of the RNA sample including the presence or absence of degradation products Contents A4 1V Index The 2100 expert software plots fluorescence intensity versus size migration time and produces an electropherogram for each sample Contents A488 V Index The data can also be displayed as a densitometry plot creating a gel like image Contents A49V Ladder PCR M PCR M PCR M PCR M PCR M PCR M PCR M PCR M PCR M PCR M POR M PCR M Index Preparing and Running an Electrophoretic Assay An electrophoretic chip run requires the following steps 1 Switch on the Agilent 2100 bioanalyzer and start the 2100 expert software See Starting 2100 Expert on page 31 2 Select an electrophoretic assay See Selecting an Electrophoretic Assay for a Chip Run on page 51 3 Prepare reagents chip and samples See Preparing Samples Reagents and Chips for Electrophoretic Assays on page 54 and the appropriate Application Note or Reagent Kit Guide 4 Load the chip into the bioanalyzer
47. assay by making changes on the following tabs Modify assay setpoints on the Assay Properties Tab Modify or enter additional chip sample and study information on the Chip Summary Tab NOTE The study description is stored in the 2100 expert system file Altering the study description of an assay will not affect the entries in the data files that were previously generated from this assay To update this information in the data files too they must be opened and the study must be assigned again For flow cytometric assays define or modify markers and regions on the Histogram Tab Single Grid View and Dot Plot Tab Single Grid View Contents A 265 V Index For electrophoretic assays define or modify flagging rules on the Result Flagging Tab 4 From the File menu select Save to save the method with the current name or Save as to save it with a new name Contents A 266 V Index Handling Chip Data Chip data xad files are automatically generated at the end of a chip run The xad files are given names that correspond to the choices you have made in the Options dialog box see How to Specify Data File Names and Directories on page 300 Modifying and saving chip data files 2100 expert allows to re open chip data files reanalyze them using different evaluation parameters and store the new results You can save modifications either to the original file File gt Save or under a new file File gt Save A
48. assay in 2100 expert just means pressing a button NOTE You can stop a chip run at any time for example if errors occurred or if you are not satisfied with the quality of the measurement results which you can observe during the chip run See Stopping a Chip Run on page 200 Starting the Chip Run When you have loaded the chip you can start the chip run 1 On the nstrument tab click the Start button Q start Contents A 196 V Index The chip run starts The Dot Plot sub tab shows single events cells as they are detected displayed as dots In the coordinate system the red and blue fluorescence intensity of each event can be read The name of the currently measured sample is displayed above the graph mixture Regipn 1 105 o Red Fluorescence gt 10 10 10 10 10 107 10 10 Blue Fluorescence Total Events 1036 of gated 52 2 Chip Setup Dot Plot Contents A 197 V Index The number of the sample that is currently being measured is indicated on the information bar Running The status bar at the bottom of the screen shows the measurement progress for the chip run and the COM port number used for data acquisition During the chip run you can do the following e View the chip data file in the Data context by clicking on the name of the Data File Method Selection a o e Start Stop Run Stop Method C s 4poptosis Demo xsy Data File C 2
49. bp 660 3 mol bp is the molecular weight of a single base pair Miniaturized laboratories on a microchip Expression used to describe lab on a chip technology Molecular separation techniques Processes such as gel electrophoresis liquid chromatography and capillary electrophoresis that can separate bimolecular organic substances from other compounds Contents A 352 V Index P PDF file PDF Portable Document Format is a file format created by Adobe Systems Incorporated that preserves all of the fonts formatting colors and graphics of any source document regardless of the software and computer platform used to create it Peak Baseline A local peak baseline is calculated for each peak For isolated peaks the local peak baseline is simply a straight line connecting the start point with the end point For peaks that are very close together an average baseline is used when the value between the peaks does not drop to the actual baseline Contents A 353 V Index FU Sample 6 35 30 isccsace lsasoodoococososd Teccence seseser tacecuce WE cccccccoe t t 62 64 66 68 70 72 74 76 s Peak Filter Width The Peak Filter Width setpoint determines the minimum amount of time that must elapse before a peak is recognized Peak Height The value at the center point of the peak minus the local baseline start value Point to Point Fit This curve fit is composed of line segments between each pa
50. calculated RNA concentration It might be convenient to pause the automatic analysis Electropherogram gt Pause Automatic Analysis until all changes are done Contents A 125 V Index Assigning Upper and Lower Marker Peaks For each DNA or protein sample the upper and lower marker peaks are assigned first and then the data is aligned so that the sample markers match the ladder markers in time allowing the size and concentration of the sample peaks to be determined RNA samples are aligned to a lower marker exclusively The first peak is assigned to be the lower marker and is then offset to match the lower marker in the ladder The upper marker is then assigned to the last peak in the sample well or to the peak nearest the ladder s upper marker See an example of assigned marker peaks below If you get unexpected peaks in the ladder analysis or find that the markers have been set incorrectly you may exclude peaks manually from the ladder or set a peak to be used as a marker Right clicking in the peak table causes a context menu to appear allowing you to do so at Export Configure Columns E Copy To Clipboard Ctrl C KR Scale to Selected Peak 4 Manually Set Lower Marker bey Manually Set Upper Marker Contents A 126 V Index In case the 2100 expert software did not detect the lower marker in RNA samples correctly you are able to manually assign it in the same way ae Export Configure Columns Copy
51. can be defined on the Result Flagging tab This tab is available in the Data context if an electrophoretic chip data xad file is selected and in the Assay context if an electrophoretic assay xsy file is selected General Properties Assay Properties Chip Summary Gel Electropherogram Result Flagging Log Book Rule Index Rule Comment Rule Color Ladder 2 found Found Ladder 2 Ladder 3 found PeakFound 31 ABS 1 Found Ladder 3 lt 7 Default rule TRUE All Other Samples Z Functions PeakFound PeakConcentration PeakFoundAuto Rule Condition jil NumberOfPeaks PeakFound 93 4BS 1 gt 0 Total4rea Found Ladder 2 Variables Operators Rule Comment Well Ladder 2 Found SampleName SampleCategory Rule Color 2yntax PeakFound float size enum Windowtype float Window int ChannelID Description Used to check whether certain sized fragment is found or not Tt Returns the PeakID of the peak if the fragment was Found otherwise returns 0 Size is the expected fragment size in bp nt kDa Windowtype can be PER or ABS PER User will give the window size as percentage of fragment size e g 10 ABS User will give the window size as absolute unit of peak size e g 10 base pairs bp window defines the window around the Size considered as Fragment size ChannellD This is default parameter if not provided default va
52. com chem labonachip Code Example the command line parameter port 2 User input Example Enter 50 MB Contents A12V Index Safety Notices Notes and Tips Safety notices notes and tips in this document have the following meaning WARNING A warning notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly performed or adhered to could result in personal injury or death Do not proceed beyond a warning notice until the indicated conditions are fully understood and met CAUTION A caution notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly performed or adhered to could result in damage to the product or loss of important data Do not proceed beyond a caution notice until the indicated conditions are fully understood and met NOTE A note contains important helpful or additional information TIP A tip usually points out a timesaving feature Contents A13V Index Quick Start The following step by step instructions guide you through a measurement with the Agilent 2100 bioanalyzer Preparing the Agilent 2100 Bioanalyzer 1 Ensure that the proper cartridge is installed in the bioanalyzer You can identify the installed cartridge by the number engraved at the front Note that there are also electrode cartridges without an engraved number Electrode Cartridge for electrophoretic ass
53. displays now only cells with blue and red fluorescence within the marker To evaluate this subset you can set a marker in the red histogram This second marker filters out all cells that do not have fluorescence in this range Events with high blue fluorescence Event Count Event Count Events that show both high blue and high red fluorescence i Intensity Intensity The result table see also Displaying the Results of Histogram Evaluations on page 230 of the gated histogram here the red one shows the values numerically e The total value shows the number of events that have both high blue and high red fluorescence in relation to all measured events e The of gated value shows the number of events that have high blue and high red fluorescence in relation to the blue or red events Contents A218 V Index The following image shows two histograms with a gating direction from blue to red left to right of an apoptosis assay The blue histogram shows calcein fluorescence which indicates living or dead cells high fluorescence value means living cells The red histogram shows the subpopulation of living cells with annexin V fluorescence indicating apoptosis high fluorescence value means the cell is apoptotic As a result you can see a subset of living apoptotic cells Calcein Annexin CY5 Number of 40 events M 100 30 iia ae ois Z 6 Calcein a Histogram 5 5 z 50 z 15 10
54. example e if the quality of the measurement results does not meet your expectations e if for example after three samples you already have the information you desired and you want to start another chip run NOTE You cannot resume a stopped chip run NOTE If you stop a chip run automatic export see Exporting Chip Run Data Automatically on page 280 and automatic print see How to Turn on and Configure Automatic Printing of Chip Run Reports on page 291 does not take place To stop the assay 1 Click the Stop button Stop OR Select Stop from the nstrument menu Contents Ab6 V Index NOTE Data acquisition of the current sample will be aborted The following message appears 2100 expert x x re you sure you want to stop the run 2 Click Yes to stop the chip run When the chip run is aborted you can e Switch to the Data context where you can view analyze and evaluate the results if any of your chip run see Displaying the Measurement Results Electrophoresis on page 72 and Analyzing and Evaluating the Results of an Electrophoretic Assay on page 81 e Stay in the nstrument context where you can start the next chip run for example Contents A 68 V Index Entering Chip Sample and Study Information During or after a chip run you can document the run by entering information on chip samples and study 1 In the Data context select the Chip Summary tab 2 On
55. fluorescence intensity values flow cytometric measurements are triplets of migration time red fluorescence and blue fluorescence Base assay information Because a chip run is always based on an assay file a information from the assay file becomes part of the chip data file Run log Events occurring during the chip run such as the start and end time or any errors or problems are entered in a run log which is also saved in the chip data file Evaluation information These are modifications you made during data evaluation such as modified gel coloring manually set markers manual integration modified setpoints modified result flagging rules or definitions of new markers and regions Contents A 260 V Index Comparison files You can compare the measurement results from different chip runs electrophoretic chip data files of same assay class only by collecting samples from different chip data files xad and storing them in a comparison files xac It is then possible to overlay electropherograms of these samples for example but also to compare gel like images or data tables Verification result files Verification result files xvd contain results of qualification tests regarding the bioanalyzer hardware and software The files are stored in the validation subfolder of the 2100 expert installation directory For each verification run an xvd file is generated Date and time of the verification run are in
56. icons 9 To apply the rules to your measurement results click the Apply Result Flagging icon amp If there still are syntax errors in the rule definitions an error message appears All samples are re evaluated according to the result flagging rules and displayed with the respective colors See Color Indication on page 157 for more information on the color codes Contents A 163 V Index Example Result Flagging Sample 1 contains 100 ug ml proteins The electropherogram shows 2 peaks for 2 different proteins which could be separated One peak can be found at 32 kDa LDH Sample 2 contains 60 pg ml proteins and shows 3 peaks Sample 3 contains 80 pg ml proteins and shows 5 peaks Now the following rules are defined 1 Is there a peak at 30 kDa 7 Rule 1 PeakFound 30 PER 7 2 Is the total concentration of proteins higher than 90 pg ml Rule 2 TotalConcentration gt 90 3 Were 5 to 10 peaks found Rule 3 NumberOfPeaks gt 5 AND NumberOfPeaks lt 10 Alternative Rule 3 NumberOfPeaks BETWEEN 5 10 Rule Index Rule Comment Rule Condition Rule Label Rule Color 2 3 PeakFound 30 PER 7 TotalConcentration gt 90 NumberOfPeaks gt 5 AND NumberOfPeaks lt 10 Number of peaks between 5 and 10 Any peak at 30 kDa 7 Total concentration gt 90 Default rule TRUE Applying these rules in the given order in Normal mode leads
57. including background fluorescence for example Contents A22V Index Y 2100 expert laj x Eile Context View Method Instrument Windows Help Instrument e E B oa Demo DNA 7500 Running All Instruments Ef Demo 2100 expert_DN Instrument 2 Name COM Port Jomo z Serial Instrument Instrument 3 a 2 Method Selection E Methods o Cartridge Erens Data H a Start Stop Run Stop KA Product ID Method C Demo DNA 7500 Custom xsy Ls Verification H HH Firmware Simulation Mode Data File C 2005 05 19 14 01 59 xad 1e Comparison w Method System Fluorescence N O 3 60 Time s Chip Setup Raw Signals Reading Sample 4 TTT TT Offline Demo Mode amp Mr Advanced as Advanced Operator Auto Export Auto Print The number of the sample that is currently being measured is indicated on the information bar Running The status bar at the bottom of the window shows the measurement progress for the chip run and the COM port number used for data acquisition Contents A23V Index During the chip run you can do the following e View the chip data file in the Data context by clicking on the name of the Data File COM Port Method Selection menei Yams earen Start Stop Run Stop Method C Demo DNA 7500 Custom xsy Data File Ci 2005 05 19 14 01 59 xad e Switch to any o
58. is inserted properly and the chip selector Is in the correct position 1 for electrophoretic assays 2 for flow cytometric assays For details please refer to Loading the Electrophoresis Chip into the Bioanalyzer on page 58 or Loading the Cell Chip into the Bioanalyzer on page 189 respectively Contents A20V Index c Place the chip into the receptacle The figure shows this for an electrophoresis chip The chip fits only one way Do not force it into place CAUTION Do not force the lid closed This may damage the cartridge d Carefully close the lid Electrophoretic assays the electrodes in the cartridge fit into the wells of the chip Flow cytometric assays the adapter with the gasket in the cartridge fits onto the priming well of the chip Contents A21V Index When the chip is detected the image on the nstrument tab changes to a chip E pa K a lt If the chip is not detected open and close the lid again NOTE If the AutoRun option is active the chip run starts automatically once a chip has been inserted and the lid has been closed 6 On the nstrument tab click the Start button start The chip run starts The Raw Signals sub tab shows an electropherogram of the currently measured sample The name of the sample is displayed above the graph The graph is a live plot of the migration time against fluorescence units raw data
59. marker Integrator Settings Analysis Ladder Import Setpoints Local Global Normal X Collapse General assay setpoints _ Electrophoresis properties Ladder Concentration 4 Ladder standard areas Table Concentration unit ng Sample setpoints Integrator Integration start time s 26 Integration end time s 94 Slope Threshold 0 5 Area threshold 0 5 Height threshold FU 8 Peak filter width s 0 5 Assays are stored as xsy files Contents amp Mr Advanced as Advanced Operator AMV Auto Export Auto Print Index System Context In the System context you can e define System Wide Settings for the 2100 expert software such as settings for default file names and directories signal colors or auto export functions e view the contents of the System Log Book EIE File Context Yiew Windows Help System WO B team w e Users and Roles System Wide Settings Archiving Import Export System Log Book a 2100 Expert System Wide Settings J m E baare Data File Name Run and Result Create Data file names by combining Auto Export IV Prefix 100 expert Default Export Directories VV Assay Glass IZ Serial Number VV Date Verification Time Example lt lt amp Counter 2100 expert_ASSAYCLASS_SRNUM_2005 05 19_13 38 28 Comparison Data File Directory lt gt R Default Directory C Pro
60. setpoint determines the time after the start of a run before which the last peak or fragment will be located any peaks appearing after this time are ignored In RNA assays the end time is shown on the single well display as a long dashed vertical green line With RNA assays another End Time setpoint is available that controls the end time for an individual peak Additionally individual peak end times can be moved manually by dragging the diamond shaped end points shown in the single view F Filter Width This setpoint determines the width of the polynomial in seconds to be applied to the data for filtering noise reduction The default depends on the assay selected This setting should be less than twice the width of the peaks of interest or the peaks will be distorted Peaks that are distorted by the filter have positive and negative peaks on both sides To see an example of such distortion increase the filter width to 5 Contents A 347 V Index Firmware The firmware is a program to control the hardware of the Agilent 2100 bioanalyzer It is downloaded from your computer to the Agilent 2100 bioanalyzer and controls among others data transfer or the measurement procedures Flow Cytometry A method to detect cells with certain properties In a continuous stream stained cells pass through a light beam The emitted fluorescence is used for counting and differentiation Flow Cytometry Standard FCS The FCS file format is the st
61. syringe and electrode cleaner with each new LabChip kit Do not touch the Agilent 2100 bioanalyzer during a chip run and never place it on a vibrating ground Keep all reagents and reagent mixes refrigerated at 4 C when not in use Allow all reagents and samples to equilibrate to room temperature for 30 minutes before use Use loaded chips within 5 minutes Reagents might evaporate leading to poor results RNA Assays Always wear gloves when handling RNA and use RNase free tips microfuge tubes and water It is recommended to denature all RNA samples and RNA ladder by heat before use 70 C 2 minutes Always vortex the dye concentrate for 10 seconds before preparing the gel dye mix Contents A 56 V Index Protein Assays e Store Protein sample buffer at 20 C upon arrival Keep the vial in use at 4 C to avoid freeze thaw cycles e Allow the dye concentrate to equilibrate to room temperature for 20 minutes before use to make sure the DMSO is completely thawed Protect the dye from light during that time Vortex before use e Allow all other reagents to equilibrate to room temperature for 10 minutes before use e Use 0 5 ml tubes to denature samples Using larger tubes may lead to poor results caused by evaporation Contents A5 V Index Loading the Electrophoresis Chip into the Bioanalyzer The Agilent 2100 bioanalyzer uses different cartridges for electrophoretic and flow cytometric assays For electrophore
62. the Sample Information sub tab you can enter additional information such as sample names and comments On the Study Information sub tab you can enter information such as the name of the current study the laboratory location and the experimenter for example Contents A69 V Index General Properties Assay Properties amp Chip Summary Gel Electropherogram l Result Flagging l Log Book l 3 Ea 3 g 2 E 2 7 Data File Location Created Modified Software 2100 expert_DNA 7500_00000_2005 05 19_14 01 59 xad C 2100 expert Secured4rea Data dsDNA DNA 7500 Demo DNA 7500 Custom Mai 19 2005 14 02 05 Mai 19 2005 15 03 38 Created by version B 02 01 51211 modified by B 02 01 51211 File Version 3 Latest version Sample Name Sample Comment Rest Digest Status Observation Result Label Result Color Approved 1 PCR Mix 1 25 35 50 53 70 O pa A Not Reviewed 2 PCR Mix2 150 158 200 210 O y A Not Reviewed 3 PCR Mix 3 500 550 600 650 m y A Not Reviewed 4 PCR Mix 1 25 35 50 53 70 D y A Not Reviewed 5 PCR Mix 2 150 158 200 210 m w A Not Reviewed 6 PCRMix3 500 550 600 650 m w A Not Reviewed 7 PCR Mix 1 25 35 50 53 70 m w A Not Reviewed 8 PCR Mix 2 150 158 200 210 im y A Not Reviewed 9 PCR Mix3 500 550 600 650 m ww A Not Reviewed 10 PCR
63. the mouse pointer on any corner of the selected region The mouse pointer changes its shape to a double arrow 3 Click and drag the border to the new size Upon moving the mouse pointer changes its shape to a crosshair and the borders of the region appear as dashed lines 4 Release the mouse button Contents A 238 V Index To change size and position numerically 1 Double click the region to open the Configure Region dialog box 2 Enter fluorescence values for the left right bottom and top side of the rectangle to define position and size of the region These values correspond to the upper and lower marker limits of the blue and red histograms 3 Click OK How to Insert a Region in All Dot Plots If you have defined a region for one sample you can copy it to the other samples of the assay To insert a region in all dot plots 1 Left click the region border to select the region that you want to use as source The nsert region into all dot plots button Sis enabled 2 Click this button The Copy Region dialog box appears which asks whether or not the source region should be used as reference The region will be inserted in the dot plots of all other samples When you change the properties of the region all copies of the region will also be changed 3 Click Yes to define the source region as reference 0R Contents A 239 V Index Click No to create new regions that are not connected The region will be insert
64. to a sample This lets you easily identify samples with certain properties immediately after a chip run The color assignment is carried out by applying a sequence of rules to the measurement results obtained for the sample The rules are defined on chip level and are applied to all samples of the chip Two modes are available to define result flagging rules e Form Mode In this mode you can easily compose an expression by selecting functions and operators from given lists If necessary additional attributes have to be provided By selecting a logical operator AND AND NOT OR OR NOT further terms can be combined to form a more complex expression The last term of the expression ends with the operator NONE e Editor Mode This mode is more flexible and allows you to write arbitrary complex expressions by using functions variables and operators TIP You can export result flagging rules and import rules from other assay or chip data files See Exporting Result Flagging Rules on page 285 and Importing Result Flagging Rules on page 2 6 Contents A 152 V Index Regardless of how you create the result flagging rules there are two options available for the order in which the rules are applied e In Normal mode the rules are applied in the given order and the first matching rule will determine the color of the sample All rules are applied subsequently The first rule which applies to the sample defines its color So you sh
65. 0 Gated by Calcein 0 06 742 66 930 Annexin on alle 21 99 3438 99 827 Annexin on subset 21 99 3438 99 428 Ki E A 206 V Index How to Navigate Through the Samples At any time even during a chip run you can scroll though all samples either in histogram or dot plot view To navigate through samples using the Tree View Panel 1 If the tree view is not visible select View gt Tree View The Tree View Panel appears to the left of the tabs and shows all chip data and assay files as nodes 2 Click any sample name The histogram or dot plot of the sample is shown in single view To navigate through samples using the Lower Panel 1 If the lower panel is not visible select View gt Lower panel The lower panel appears in the lower left corner showing a chip icon 2 Click any well on the chip icon To browse through samples 1 From the Histogram or Dot Plot menu select Next Sample or Previous Sample 0R Click the Next Sample or Previous Sample button in the histogram dot plot toolbar To switch between histogram and dot plot view 1 Click the Histogram or Dot Plot tab to display the results of the selected sample as a histogram or dot plot Contents A 207 V Index How to Change the Display of Histograms and Dot Plots In single view it is possible to change the display of histograms and dot plots In histograms and dot plots you can e zoom enlarge or reduce using the mouse the graphs to di
66. 005 04 27 _13 40 03 xad e Evaluate any chip data file in the Data context e Compare samples in the Comparison context e f necessary abort the chip run by clicking on the Stop button see also Stopping a Chip Run on page 200 All data that was collected up to the stop point will be saved Contents A 198 V Index After the chip run is completed you can e Switch to the Data context where you can view analyze and evaluate the results of your chip run see Displaying the Measurement Results Flow Cytometry on page 205 and Analyzing and Evaluating the Results of a Flow Cytometric Assay on page 211 e Stay in the nstrument context where you can start a new chip run for example Contents A 199 V Index Stopping a Chip Run You can stop a chip run at any time for example e if the quality of the measurement results does not meet your expectations e if for example after three samples you already have the information you desired and you want to start another chip run NOTE You cannot resume a stopped chip run NOTE If you stop a chip run automatic export see Exporting Chip Run Data Automatically on page 280 and automatic print see How to Turn on and Configure Automatic Printing of Chip Run Reports on page 291 does not take place To stop the assay 1 Click the Stop button Stop OR Select Stop from the nstrument menu Contents A 200 V Index NOTE Data acquisiti
67. 4 s Sample 5 Ka C Sample 6 Bors O Sample 7 Verification Sample 8 LE O Sample 9 SA C Sample 10 C Sample 11 Comparison Sample 12 CS C Ladder Method System 50 300 500 700 1000 2000 10380 bp 4 gt Size bp Conc ng l Molarity nmolff Observations a 1 50 8 30 251 5 Lower Marker 2 74 8 80 179 7 3 113 1 92 2 7 ee ee 4 165 0 95 8 7 S_ _ _ EE 215 2 56 18 0 i _ i 261 1 55 9 0 i ie 310 1 98 9 7 E 8 417 2 94 10 7 oe ed ee ee A 514 7 66 22 6 T l L illz 456789 101112 Results Peak Table Legend Errors amp Mr Advanced as Advanced Operator Auto Export Auto Print Peaks have automatically been detected and their characteristics such as size concentration purity or molarity have been calculated and are shown in the Peak Table at the bottom of the window Contents A260 V Index What You Can do When the Measurement is Finished When the measurement is finished you can Document your chip run by entering sample names chip comments and study information for example Evaluate the measurement results by analyzing gel like images and electropherograms electrophoretic assays or histograms and dot plots flow cytometric assays Analyzing and Evaluating the Results of an Electrophoretic Assay on page 81 Anal
68. 6 s Contents A 340 V Index The figure below shows baselines established for Total RNA assay fragments Total RNA fragments are determined on a peak by peak basis and an overall baseline is shown from the start to end time FU Sample 7 70 60 50 40 Sample 7 20 Contents A 341V Index The figure below shows baselines established for an mRNA assay mRNA fragments are determined on a peak by peak basis and an overall baseline is shown from the start to end time FU Sample 3 Contents A 342 V Index NOTE With RNA assays you can move the lines marking the start and end points for data analysis shown by the long dashed vertical green lines which will adjust the entire baseline for calculation of the area of the total sample Baseline Plateau This setpoint found in the setpoint explorer rejects brief low slope areas such as at peaks and between non baseline resolved peaks The signal is recognized to be at baseline whenever the slope of the data is less than the Slope Threshold setpoint either positive or negative for longer than the time set for the Baseline Plateau BMP file BMP is the standard Windows image format The BMP format supports RGB indexed color grayscale and bitmap color modes Bubble If the tip of a pipette is not positioned all the way to the bottom of a well bubbles can result and sometimes bubbles happen even when you are very careful The vortexing
69. 8 V Index To show the setpoint explorer on the E ectropherogram Gel tab click the vertical bar on the right edge of the application window The setpoint explorer appears For electrophoretic assays you can modify the setpoints e globally that is for all samples Global tab e locally for the current sample Local tab Click the nodes to expand and the nodes to collapse branches Setpoints that you can change are white To edit a setpoint double click the value enter the new value and press enter They are applied automatically Contents A119 V Index Local Global Advanced X Collapse Z Quantitation aj Concentration of upper 4 2 Concentration of lower 8 3 Sizing Standard Curve Smear Analysis Perform Smear Analysis Baseline calculation Baseline start time s Baseline end time s Zero Baseline Z Filter Settings Filter width s Baseline correction Point to Point 26 94 Perform Baseline Corre O Integrator Tnkanratinn chart Firma lel mm When you try to change any global setpoints where local settings have been applied the software prompts you as to whether you want to overwrite the local custom settings If you decide to overwrite the custom sample settings all local settings you made will be discarded If you decide not to overwrite the custom sample settings the global settings will not be applied where local settings have been chan
70. Export M Result Tables Create a csv file containing result table values IV Sample Data Export textual list files one file per sample M FCS Data Create FCS standard files one file per sample IV Dot Plot Images T Formats Export the Dot Plot image one file per sample IV Histogram Images T Formats Export the blue and red histogram images one file per sample 3 Activate the Auto Export check box if you want a data file to be exported automatically after every chip run Contents A 305 V Index 4 Inthe Electrophoresis Export section specify which elements are to be included in the exported file for electrophoresis measurements 5 Inthe Flow Cytometry Export section specify which elements are to be included in the exported file for flow cytometry measurements 6 Select Default Export Directories in the tree navigation and define the default directories for the various file types Optionally you can activate the Create daily subdirectories check box to automatically export the files of each day to separate directories How to Activate Software Licenses By installing the 2100 expert software you have also installed a license administration tool This tool is used to activate the different software modules The following licenses can be ordered separately e 2100 electrophoresis license e 2100 flow cytometry license e 2100 security pack license e 2100 instrument control license Contents A 306 V Index To activate a
71. File All Comparison Files Comparison Summary Gel E ComparisonFile0 Protei C IgG non reduced LI IgG nonreduced C IgG non yaducos O Ladder 8 lt Delete Sample from SES File ol IgG reduced C Sample 1 Contents A 147 V Index 7 When you have added all your samples you can select the Comparison Summary Tab which displays information on the comparison file and lets you enter a comment regarding the comparison All Comparison Files L IgG non reduced L IgG nonreduced L IgG non reduced L Ladder L IgG reduced L Sample 1 Select Data Files Contents ComparisonFileO Protei Gel Electropherogram Comparison Summary File ComparisonFile0 Protein 200 xac Location Assay Class Protein 200 Created November 05 2003 11 44 45 4M Modified November 05 2003 11 44 45 4M Software Created by version B 01 01 51116 modified by B 01 File Data 2003 11 05 2100 expert_Protein 200_0000 1 Data 2003 11 0512100 expert_Protein 200_0000 Data 2003 11 05 2100 expert_Protein 200_0000 1 Data 2003 11 05 2100 expert_Protein 200_0000 Data 2003 11 0512100 expert_Protein 200_0000 100 expert data 2100 expert_Protein 200_0000C A 148 V Index 8 To compare the electropherograms of samples go to the E ectropherogram tab click Overlaid Samples in the toolbar and select the samples to be compared E
72. Is inserted Adjust the regions by directly moving the dashed lines in the electropherogram To remove a region right click the dashed line in the electropherogram and select Remove Region from the context menu NOTE The smear analysis table can be directly edited by selecting the region table under Smear Analysis in the setpoint explorer Contents A 114V Index Local Global advanced hed Collapse Alignment Align to upper marker x Align to lower marker x Quantitation Concentration of upper ma 4 2 Concentration of lower ma 8 3 Sizing Standard Curve Point to Point Smear Analysis Perform Smear Analysis Regions ql i N Baseline calculation Baseline start time s 26 Baseline end time s 94 Filter Settings Filter width s 0 5 Polynomial order 4 Raceline correction In the smear analysis table you can edit the Region Start Size and Region End Size for example Smear Regions eee Cancel Contents A 115 V Index Changing the Data Analysis Different sets of parameters data analysis setpoints can be changed in the software in order to modify the data evaluation for sample analysis e Filtering parameters e Peak find parameters for all samples peak height for individual samples e Enabling smear analysis e Align to upper and or lower marker e Adding deleting ribosomal fragments for RNA assays only e Manual integra
73. Manual Integration 2 Re 0 0 0 0 0 0 3 9 5 0 0 0 0 0 0 System Peak p gt 61 9 471 4 453 4 100 0 Calibration Pr 5 gt 210 0 100 0 0 0 0 0 Upper Marker Contents A134 V Index 3 Right click a baseline point and select Remove Peak from the context menu The two baseline points and the connecting line will disappear and the integration results shown in the result and peak tables will be updated Size kDa Rel Conc ug ml Calib Conc ug ml Total Observations 1 4 6 0 0 0 0 0 0 0 Lower Marker zm 8 3 0 0 0 0 0 0 System Peak 3 9 5 0 0 0 0 0 O 5ystem Peak gt 210 0 100 0 0 0 0 0 Upper Marker Contents A 135 Y Index Example Inserting peak baselines To insert peaks manually 1 Highlight the E ectropherogram tab in the Data context and zoom into the electropherogram to enlarge the peak of interest Size kDa Rel Conc pg m Calib Conc ug ml Total Observations 1 4 6 0 0 0 0 0 0 0 Lower Marker 2 8 3 0 0 0 0 0 0 System Peak 3 9 5 0 0 0 0 0 0 System Peak gt gt 210 0 100 0 0 0 0 0 Upper Marker Contents A 136 V Index 2 Right click the electropherogram and select Add Peak from the context menu Sample 3 J gt Undo Zoom iS Undo all ig Copy Electropherogram P Save Electropherogram Size kDa Rel Conc pg m Calib Conc ug ml 6 0 0 0 0 0 0 0 0
74. RNA ladder containing a mixture of RNA of known concentration is run first see the electropherogram below The concentrations and sizes of the individual base pairs are preset in the assay and cannot be changed Electropherogram of RNA 6000 Ladder Ambion Inc cat no 7152 Contents A8 V Index NOTE Peak ratios for the RNA ladder may vary from one batch of RNA 6000 ladder to the next Assay performance will not be affected by this variation 5 For the Eukaryote or Prokaryote Total RNA assay the RNA fragments either 18S and 28S for eukaryotic RNA or 16S and 23S for prokaryotic RNA are detected After detection the ratio of the fragment areas is calculated and displayed FL Sampe 12 lower marker 6 To calculate the concentration of the RNA the area under the entire RNA electropherogram is determined The ladder which provides the concentration area ratio is applied to transform the area values into concentration values Contents A 88 V Index Alignment of RNA Samples The marker solution that is part of each RNA LabChip kit contains a 50 bp DNA fragment This fragment is used as lower marker to align all samples By default the RNA alignment and the subtraction of the lower marker are enabled for RNA Nano assays The marker is displayed as the first peak in the electropherogram Contents A89 V Index The RNA Integrity Number RIN The RNA integrity number RIN is a tool designed to help scientists
75. Software Preparing the Agilent 2100 Bioanalyzer 1 Ensure that the proper cartridge is installed in the bioanalyzer You can identify the installed cartridge by the number engraved in its front Engraved number EHE hfe Click here to go to the table of contents Click here to go to the index Here is the current page number A Displays the previous page W Displays the next page Contents A10V Index After you have chosen a topic with the bookmarks use the buttons in Acrobat Reader s toolbar to move around within the topic Displays the next page Returns to the previous view Click several times to undo Displays the previous aa more view changes SABS AA AM EM 4 e D ii lop TOAE S Returns to the next view Click several times to redo more view changes Displays the first page Displays the last page Contents Al1lV Index Layout Conventions The following typographic conventions are used in this manual Highlight Italic Blue Courier Courier bold On screen element Example the OK button Emphasis Example Right click the Term Example Dot plots show events as dots Reference to another document Example Refer to the Agilent 2100 Bioanalyzer Troubleshooting and Maintenance Guide Cross reference or hyperlink Examples Introduction to the Key Features of the 2100 expert on page 29 http www agilent
76. Spare parts 334 Staining cells 168 Starting an assay 64 196 Stop assay 67 200 System Log 311 System verification 325 SYTO16 168 U Undo zoom 77 209 A 365 V Index Z Zoom Dot Plot 76 208 Histogram 76 208 Undo 77 209 Contents A 366 V Index
77. XML is the Extensible Markup Language a system for defining specialized markup languages that are used to transmit formatted data XML is conceptually related to HTML but XML is not itself a markup language Rather it is a metalanguage a language used to create other specialized languages 2100 expert uses the XML format to e export chip data e save and load result flagging rules XSY file 2100 expert assay file The files contain the assay properties data acquisition settings and information on chip samples and study Contents A 359 V Index XVD file 2100 expert verification result file The files contain results of verification tests regarding the bioanalyzer hardware and software xvd files are stored in the validation subfolder of the 2100 expert installation directory For each verification run an xvd file is generated Date and time of the verification run are included in the file name Example Verification_ 23 05 2005 10 28 40 Contents A 360 V Index Z Zero Baseline All electropherograms produced with the bioanalyzer show some amount of background fluorescence By default the 2700 expert software enables the zero baseline function Enabling this setting offsets the graphs shown for the individual wells but does not affect analysis The mean of 100 points before the baseline time derived when calculating well noise is used as the zero baseline value Fu IgG reduced IgG reduced
78. ag the header cell to the desired position While dragging a green arrow indicates the target position Fragment tion Molarity Observations MigrationTime i ratio ine 4 20 0 76 472 30 3 Release the mouse button The column has moved to its new position Area FragmentSize MigrationTime Concentration Molarity Observations 50 29 15 00 44 14 4 z 424 a CalculatedLowerMarker a ma sa os 24 13 40 500 66 95 53 0 ie 84 13 1500 00 115 56 2 10 2 12 CalculatedUpperMarker Contents A 296 V Index Changing the Table Height You can customize the view by changing the height of the table To increase or reduce the table height 1 Position the mouse pointer above the heading row of the table and move it upwards until the cursor s shape changes to a double arrow 2 Click and hold the left mouse button and drag up or down 2 12 CalculatedUpperMarker Results Peak Table 3 Release the mouse button Contents A 297 V Index In this example the Peak Table freed screen space for the gel like image above the table Contents A298 V Index Administering System Functions The 2100 expert software provides the following configuration options and system functions e Default data file names and directories can be specified Also settings such as for automatic printing or automatic data export can be set up See Configuring 2100 expe
79. alcein Calcein Calcein Calcein Chip Lot Reagent Kit Lot 12 2556 Chip Comments by Ellen Senge Information You may find some input fields already filled in because chip sample and study information are taken over from the base assay or chip data file Contents A 203 V Index 3 From the File menu select Save TIP You can import chip sample and study information from txt or csv files This is especially helpful and time saving if you already have documented a similar chip run in another chip data file Refer to Importing Chip Sample and Study Information on page 275 for details Contents A204 V Index Displaying the Measurement Results Flow Cytometry You can view the measurement results of a flow cytometric chip run as histograms or dot plots e You can display the histograms dot plots either one sample at a time or all samples at the same time to get an overview of the chip run for example to see the progress of a reaction See How to Switch Between Single View and Grid View on page 206 e You can navigate through the samples See How to Navigate Through the Samples on page 207 e You can change the display of histograms and dot plots to make details better visible See How to Change the Display of Histograms and Dot Plots on page 208 Contents A 205 V Index How to Switch Between Single View and Grid View To switch between single view and grid view
80. allation Qualification Test Abort Continue 10 After all tests have been executed the following message appears 2100 expert xi Laj J validation Run Complete 11 Click OK Contents A 330 V Index 12The Status column shows which of the tests have been run successfully which have failed and which have mixed results with multiple executions of Executed passed J ects nce ANEx a a a tf Executed passed i ae ee 2 a tl Executed passed i Contents A331 V7 Index 13 To view details on test execution select the Results tab Configuration Results LogBook Test Name Installation Qualification Test Execution Date and Time Status Comment v _ 27 04 2005 16 20 25 x Failed failed results beyond limits Name Installation Qualification Test Description Verifies that files and configurations have been installed to their appropriate location and display correct attributes Approximate Time 300s Test Status Executed failed IQ SW results Installation Qualification of Agilent 2100 Bioanalyzer System Software 2100 expert Version 27 04 2005 16 20 28 Check of application directory structure Passed C Programme Agilent 2100 bioanalyzer 2100 expert help Passed C Programme Agilent 2100 bioanalyzer 2100 expert configuration validation OQ electrophoresis Passed C Programme Agilent 2100 bioanalyzer 2100 expert reports templates Passed C Pro
81. amount of stain bound to the cell and therefore a specific cell property and the physical properties of the stain itself The Agilent 2100 bioanalyzer lets you determine the number of cells characterized by a specific pattern of fluorescence For example to differentiate between dead and living cells you can use a non fluorescent dye that becomes fluorescent when metabolized by living cells After staining with such a dye living cells have a higher fluorescence value than dead cells The second dye could bind to a specific surface marker on a subpopulation of the cells This allows you to determine the number of living cells that contain your marker of interest For evaluation 2100 expert displays the results as histograms and as dot plots Contents A 171V Index Generating Histograms 2100 expert counts the events sorts them and displays them according to their fluorescence intensity in histograms For each color measured a histogram displays the number of events related to the fluorescence intensity A large number of events with a high fluorescence value means that a large number of cells containing the fluorescence dye were detected In the following illustration cells which fluoresce in both colors are highlighted As a Cell passes through the detector its blue and red fluorescence values are measured and the count is increased for both channels at the appropriate intensity Evert Court Illustrates a cell that
82. ample information Assay details complete list of data analysis setpoints Run Logbook Signature Logbook Audit Trail For flow cytometric chip data files xad you can include Dot plot summary all regions shown in an overview Dot plot statistics all statistical data of the result table Histogram summary all histograms shown in an overview Histogram statistics all statistical data of the blue and red histograms result tables For electrophoretic chip data files xad depending on the assay type you can include Electropherograms Gel like image Result tables Standard curve Calibration curve Contents A 287 V Index To print a report 1 Switch to the Data context 2 Inthe Tree View Panel select the chip data xad file you want to generate a report of 3 From the File menu select Print Depending on the file type different dialog boxes appear m Print Item Run Summary Dot Plot Summary JV Histogram Summary I Run Logbook IV Assay Details JY Dot Plot Statistics JV Histogram Statistics JV Signature Logbook I Audit Trail gt Samples All Samples Samples Enter sample number and or sample ranges separated by commas Example 1 2 3 6 Options 6 per page x m Save To File PDE FilePath C i pert Apoptosis I HTML File Path fc 13 40 03 html Preview Cancel Page Setup
83. and select Add Sample to New Comparison File EF 2003 11 05_11 23 53 xad Contents A 14V Index A new comparison file appears in the upper part of the tree view containing the sample The sample is selected and its electropherogram is shown Electropherogram IgG non reduced All Comparison Files K ComparisonFile0 Protei L IgG non reduced Comparison Summary Gel Electropherogram IgG non reduced IgG non reduced Note that the E ectropherogram Tab Single Grid View has the same functionality as in the Data context Contents A 145 Y Index 5 You can now add further samples from any of the open xad files to the comparison file v Select Data Files E 2003 11 05_11 23 53 xad Size kDa 6 0 Add Sample to Compari rc File Add Sample to New Comparison File D Results Peak Table i TIP Double clicking a sample name in the ower part of the tree view or dragging a sample name into the tree view adds the sample to the comparison file that is currently selected in the upper part of the tree view Or if no comparison file is selected creates a new comparison file and adds the sample to it You are notified if you try to add a sample of a xad file that has the wrong assay type Contents A 146 V Index 6 You can also remove samples from a comparison file Right click the sample name and select Delete Sample from Comparison
84. andard format used in flow cytometry to exchange data between several applications G GIF file Graphics Interchange Format GIF is a graphics file format that uses a compression scheme originally developed by CompuServe Because GIF files are compressed the file can be quickly and easily transmitted over a network This is why it is the most commonly used graphics format on the World Wide Web H Height Threshold The Height Threshold setpoint determines whether a peak is kept It represents the minimal peak height For each peak the difference between the start point value and the Contents A348 V Index center point value local baseline must be greater than the Height Threshold value This setting is chosen in the setpoint explorer Histogram Histograms are bar charts to display for example a frequency distribution HTML file HTML Hyper Text Markup Language is the authoring language used to create documents on the World Wide Web HTML defines the page structure fonts graphic elements and hypertext links to other documents on the Web J JPG file Joint Photographic Experts Group Image File A JPEG file is a compressed raster or bitmapped graphic image When a JPEG is created a range of compression qualities may be considered JPEG compression is a lossy process which means that you sacrifice quality for file size the more you compress the image the highest quality images results in the largest file size Whereas
85. are tests can be run for e Installation verification e System verification Contents A 38 V Index Verification results are automatically saved in xvd files You can re open xvd files to review verification results For details refer to Performing Verifications on page 325 Contents A39 V Index Comparison Context In the Comparison context you can open multiple electrophoretic chip data files and compare samples of the same assay class DNA 1000 for example It is possible to overlay electropherograms recorded by the bioanalyzer and compare the results 382100 expert C ent 2100 bioanalyzer 2100 expert SecuredArea Comparison 2005 05 19 Comparisonfile1 DNA 500 xac Eile Context View Electropherogram Windows Help Comparison Ieho E amp oo me R ECES Overlaid Samples contexts Electropherogram PCR Mix 3a E a Comparison Files Comparison Summary Gel Electropherogram Comparison Log Book Fi mj Instrument FU PCR Mix 3a 10004 Ez Verification Comparison SS 5004 System i 4 30 40 50 60 70 80 90 s 2 iewport 2 PCR Mix 3a PCR Mix 1 Legend Errors Mr Advanced as Advanced Operator Auto Export Auto Print Comparison results can be saved in xac files You can re open xac files to review the comparison results and to add further samples for comparison Contents A40V Index Assay Context In the Assay cont
86. astcesssauetadootehecoactencstatacassadzsossn adie deandecentuteceecedeaten 8 How to Use this Manual icc cesses cate acetates Decaasene lana ddan neta awesteecaaeneans 10 OH ne 9 te ee neem re Ban Ser ener ene E ee arene eee ee 14 Looking at 2100 Expert Reve sees or een near eT nme ENR Mon ry BOER OMS pte oe UeNUenPR RCo 28 Introduction to the Key Features of the 2100 expert cccccccsececsessseessssescseeesseeeenees 29 Starting 2100 EX BE gsc cast ecscdcadesacedSuacececesciciosatastonc dkcstaansdcanctasdedeensdveustavasendesersadeceacexsneacnareces 31 2100 Expert Work Area occirioceceteasheisiceteixecenntstatesinsextest eens ade tndiee ieee aaa adea 32 Closing 2100 EXPE g eaeeearee nee ene eeen mere noe eet a Tn 43 Running and Evaluating Electrophoretic Assays s csssscsssssscssseseeseceesessecseenseanens 45 Principles of Nucleic Acid and Protein Analysis on a Chip cccccccsssseecseseseseeeees 46 Preparing and Running an Electrophoretic ASSa cccccscsccsssseessesescsceseeeecseseseseseeeees 50 Analyzing and Evaluating the Results of an Electrophoretic Assay cccccssseeeee 81 Pe ST Fla goin ieran E EE 152 Running and Evaluating Flow Cytometric AssayS ssssssssssssnensnsennunnnnnnnunnnnnnnnnnnnnnnnnnns 166 Principles of Flow Cytometric Measurements cccccccsescscsesssscecsesescseseseececsescseseseees 167 Overview of Flow Cytometric Assays ccccscscscsescsessesscessescscsesesecsescscsesesesesess
87. at closed position Contents A 60 V Index To load the prepared chip into the Agilent 2100 bioanalyzer 1 Open the lid and remove any chip 2 Adjust the chip selector to position 1 as shown in the following figure To avoid using incompatible chips and cartridges a chip selector is installed in the bioanalyzer This ensures that the chip matches to the installed cartridge Move chip selector in These steps are required for inserting DNA RNA and Protein chips in the bioanalyzer Contents A61V Index CAUTION Do not force the chip selector handle when a chip is inserted in the bioanalyzer 3 Place the prepared chip into the receptacle The chip fits only one way Do not force it into place Chip selector in position 1 4 Carefully close the lid CAUTION Do not force the lid closed This may damage the cartridge If the lid does not close completely check that the cartridge and chip are inserted properly and the chip selector is in the correct position Contents A62V Index When the chip is detected the image on the nstrument tab changes to a chip s 2 o gt lt If the chip is not detected open and close the lid again NOTE The displayed image depends on the assay selcted in the software not the type of chip inserted If you would like to run a DNA chip but a protein chip appears you have selected the wrong assay
88. ay for a Chip Run on page 181 Prepare chip and samples Refer to Preparing Samples and Chips for Flow Cytometric Assays on page 185 and to the appropriate Application Note and Reagent Kit Guide Load the chip into the bioanalyzer For details refer to Loading the Cell Chip into the Bioanalyzer on page 189 Start the chip run This is described in Running a Flow Cytometric Assay on page 196 When the chip run has finished you can e Have a first look at the results see Displaying the Measurement Results Flow Cytometry on page 205 e Document the chip run see Entering Chip Sample and Study Information on page 202 Contents A 179V Index e Analyze and evaluate the results Using Histograms for Evaluation on page 212 Using Dot Plots for Evaluation on page 233 Evaluating Antibody Staining Apoptosis and GFP Assays on page 242 Contents A 180 V Index Selecting a Flow Cytometric Assay for a Chip Run To select an assay 1 Switch to the nstrument context 2 Inthe Tree View Panel select the bioanalyzer you want to use g All Instruments y Instrument 1 v In the upper left of the nstrument tab an icon shows the status of the bioanalyzer You should see one of the following icons lid open closed indicating that the bioanalyzer is detected by the system 3 If you do not see one of these icons check that the bioanalyzer is switche
89. ays Pressure Cartridge for flow cytometric assays Contents Al4V Index 2 If you have to change the cartridge follow the instructions in Loading the Electrophoresis Chip into the Bioanalyzer on page 58 or Loading the Cell Chip into the Bioanalyzer on page 189 respectively Switching on the Agilent 2100 Bioanalyzer 1 Make sure the bioanalyzer is connected to line power and connected to the PC 2 Turn on the line switch at the rear of the instrument The status LED at the front of the bioanalyzer should light up Lid Status LED Contents A15V Index The status LED shows you the current status of the instrument Signal Meaning Green light Instrument is switched on and ready for measurement Green blinking Measurement is running Orange blinking Instrument is busy running self diagnostic for example Red light Instrument is not ready for measurement Switch the instrument off and on again If the problem persists call Agilent service Running a Measurement 1 To start the 2100 expert software on the connected PC go to your desktop and double click the following icon 2100 ma Contents A16V Index After startup of the software you enter the nstrument context 2100 expert F a x Eile Context View Method Instrument Windows Help Instrument g e E amp oa Instrument Data G Verification All Instruments Instrument Diagnostics K Demo Instrume
90. ays Antibody staining lets you measure protein expression on the surface or inside a cell by means of specific antibodies Either the primary antibody itself is conjugated with a dye or you must use a labeled secondary antibody that recognizes the primary antibody When you measure the fluorescence of the cells you can compare the relative expression of protein in individual cells and use this information for population analysis Typically you can use a red dye such as APC Allophycocyanin or Cy5 to measure antibody presence You can use a blue dye like calcein to detect whether or not the cells are living or like SYTO 16 to stain the nucleic acids of all cells For detailed information refer to the application note Detecting Cell Surface and Intracellular Proteins with the Agilent 2100 Bioanalyzer by Antibody Staining For a detailed description on how to evaluate the results using markers and regions refer to Using Histograms for Evaluation on page 212 and to Using Dot Plots for Evaluation on page 233 Gating direction The gating direction is from blue fluorescence to red fluorescence Depending on the dye you use you should use all cells nucleic acid dye or only living cells calcein living dyes for gating Contents A243 V7 Index Histogram Evaluation The blue histogram is used for gating High fluorescence in the blue histogram means that the cells are living if a life indicating dye is used Low fluorescence m
91. ber is shown on the Results sub tab of the Gel or Electropherogram tab of the Data context It is also included in XML export files and in printed reports Assay Properties Chip Summary Gel Electropherogram Result Flagging Log Book Sample 3 RNA Area 61 2 RNA Concentration 101 ngul rRNA Ratio 28s 18s 1 2 RNA Integrity Number RIN 7 9 j Peak Table Fragment Table Legend Errors Contents A91V Index NOTE Until now the computation of the RIN has only been validated for eukaryote total RNA Nano samples The 2100 expert software also calculates the RIN for prokaryote total RNA samples and for the RNA 6000 Pico assay Be aware that for these samples the RIN has not been validated in extensive downstream experiments Although the lower quantitative limit of the RNA 6000 Nano assay is specified as 25 ng ul it is recommended to use at least 50 ng pl for a meaningful RNA integrity number When using lower concentrations higher sample to sample variances of the RIN may be observed Examples for RNA Integrity Numbers A database of about 1300 mammalian total RNA samples was created using the RNA 6000 Nano assay The samples came from different species mainly human rat and mouse tissues preparation methods concentrations and degradation states All samples were classified according to their degradation state Numbers from 1 to 10 were used as labels 10 stands for a p
92. box and click the Settings button next to this check box The Auto Print dialog box appears M Print Item JV Assay Details JV Sample Data IV Run Summary For Flow Cytometry data 2 per page zi Save To File J PDF C alyzer 2100 expert Data data file name gt pdf Fp V HTML Ci lyzer 2100 expert Datal lt data file name gt html a Page Setup Printer r i Cancel Help NOTE The Auto Print settings are independent from those made via the Print command of the File menu 4 Adjust the settings Contents A 291V Index Inthe Print Item section select the options that are to be included in the report Inthe Save To File section you can redirect the automatic printouts to pdf and html files Note that no print output is generated if you select the PDF and or HTML option Using the Page Setup and Printer buttons you can access system dialog boxes allowing you to select a printer for the automatic print and specify the print medium and page layout 5 Click OK to confirm the automatic print settings Contents A292 V Index Configuring Tables 2100 expert uses various tables to present data e Result tables e Peak tables e Fragment tables e Log book tables In some cases you might want to reorganize the way the data Is presented To do so you can hide or show columns change the column sequence and adapt the table height Area Fra
93. ce and offset are printed on 1 the chip Can be used multiple times Electrode Diode Test Chip Can be used multiple times 1 Test Chip Kit for Flow Cytometry Assays reorder no G2938 68200 Test Chip Comment Quantity Cell Autofocus Test Chip Required for Pressure Control Test System 1 Leakage Test and Optical Drive Test Can be used multiple times Contents A 320 V Index How to Run Instrument Diagnostics Tests NOTE Diagnostics tests cannot be run while the 2100 expert software is performing a chip run To run diagnostics tests 1 Switch to the nstrument context 2 Inthe Tree View Panel select the bioanalyzer on which you want to run the tests 3 Switch to the Diagnostics tab All available tests are displayed in the Available Tests list The tests that can be executed depend on the type of cartridge that is installed in the bioanalyzer The 2100 expert software will generate an error message if a wrong cartridge type is detected for the selected assay To run the selected test please insert the requested cartridge type see Loading the Electrophoresis Chip into the Bioanalyzer on page 58 or Loading the Cell Chip into the Bioanalyzer on page 189 respectively Contents A321 V7 Index 4 Select the tests you want to run Select the Apply check boxes to select single tests Click Select All to select all available tests Click Unselect All to deselect all tests
94. cecssaeeeseees 176 Contents A3V Index Preparing and Running a Flow Cytometric Assay ccccccscccsssssscecsesescseseeseeecseeeseseseees 179 Analyzing and Evaluating the Results of a Flow Cytometric Assay ccccccceeeee 211 Working with Chip Data and AssayS ccccccscsssseseseeseeseseesseesaesesseessessessesseeseeseseeseas 258 2100 Expert Datel VETVIEW x xcscctccsesdacsccecetetdesastacsesedeestievischeucdechesedsseacatieeachddcstnisaceagesseaneanets 259 Handling ANS SAY Skree Aes acer ccrdn dint Taaa eee stein eeideeedes 262 Handling Chip Datassist a a a 267 Organizing Backing up and Archiving 2100 Expert Data ccecceeceeseseseseseeseeeeees 269 Importing Data e esensenesnseesnnsnsensnsansnrsrneinnrsnnsrnnennrsnsnrsntnnnsrsnnntninnrnitnnnatntnitnnnnnsrnnsnnnnnnnenn 271 DAOA LATO E DIE 1 i O 277 Printing Reports cacscsccccsessscsaccccsecsassossasdedeaccedeacssasacsadscsasece cavaceassastadeedebsesdsanseneahsentacuasnucsasneceds 286 Configuring Tables set tc eden Dnt acceler dead ea ceases ees esas oes 293 Administering System Functions cccssssssssscessesssesessecsesessecsecsesaeseesesesessesnensensans 299 Configuring 2100 GHG asais cessciacasncccdencseataechansiarstitatecstdecteaacsteade nasied ies acelin eee 300 Using Log HE OO cst acess cee cece tect casesc ze decscetastecack toss kandara niaka ssc desscose cacti ocseseseeteonsed 310 Running Instrument Diagnostics ccccssssesesecsee
95. cluded in the file name Example Verification_ 23 05 2005 10 28 40 xvd Diagnostics result files To ensure proper functioning of the bioanalyzer hardware you should run hardware diagnostics tests on a regular basis The results of these hardware tests are stored in diagnostics results files xdy in the diagnosis subfolder of the 2100 expert installation directory Result flagging rule files You can export and import result flagging rules from other assay or chip data files Result flagging rules are stored in xml files Contents A261 V7 Index Handling Assays Predefined Assays Predefined assays are provided with 2100 expert They are meant and prepared for measurements using the available LabChip kits Predefined assays such as Apoptosis or DNA 1000 are write protected Although you can open predefined xsy files and edit some of their properties you cannot save any changes under the original file name Custom Assays You can derive your own assays from the predefined assays as described in How to Create a Custom Assay on page 264 The main benefit of custom assays is that you have to do the following only once in the assay file instead of doing it again and again in the chip data files e Modify assay setpoints data analysis setpoints e Enter information on chip samples and study For example if your sample names are to be the same for a series of chip runs e Define rules for result flagging e
96. curedArealMethod Demo TemplatesiRNADemo MRNA Pira vsi Title Demo mRNA Pico Version 1 0 Ladder Concentration 1000 00 Start Time 15 00 End Time 84 00 Sizing Accuracy 15 Quantitation Accuracy 30 Assay Comments Copyright 2003 Agilent Technologies Chip Information Chip Lot Reagent Kit Lot Chip Gorments Sample 1 Sample 2 Sample 3 Fu Fu Ful 54 5 24 o 0 0 T T T T T T T T T T T T T T 2 5 x 5 4a s 2 25 30 35 sf 2 2 3 35 4 s Sample 4 Sample S Sample 6 Fu Fu Fu 44 24 5 a 0 0 0 2 23 3 35 4 f 2 5 amp 3 3 5 4 j 2 3 amp 3 3 35 4 f Sample Sample 8 Sample 9 Fu Fu Ful 44 24 s 24 14 0 0 A T T T T T T T T T T T T T 2 23 3 35 4 f 2 23 32 35 j 2 23 3 35 4 j Sample 10 Sample 11 FU Fu 10 re 5 24 o 8 T T T T T T T T T 2 23 32 35 4 f 2 32 35 j 2100 expert B 02 01 51211 Copyright 2003 2005 Agilent Technologies Inc Printed 17 05 2005 16 07 10 Contents A 290 V The following example shows the Run Summary part of an RNA chip run report Index How to Turn on and Configure Automatic Printing of Chip Run Reports A report can be automatically printed on a printer or generated as a file at the end of each chip run Saving reports as files can be helpful for documentation purposes To enable and configure automatic printing 1 Switch to the System context 2 Select Run and Result in the tree navigation 3 Activate the Auto Print check
97. curity pack that needs to be ordered separately as G2949CA and is then activated with a license key This security pack activates user management functions and electronic signature to meet the Food and Drug Administation FDA requirements 21 CFR Part 11 2100 expert provides detailed installation verification and system verification tests on both the bioanalyzer hardware and software 2100 expert allows having multiple chip data and or assay files open at the same time 2100 expert features a new integrated data evaluation tool Comparison context allowing comparison of measurement results of same assay class from different chips directly A separate data evaluation tool is no longer necessary 2100 expert features improved integration including manual integration available for DNA and Protein assays only 2100 expert allows color coded result flagging with pre defined or custom result flagging rules Flagging rules can be applied to measurement results 2100 expert now has customizable result tables and gel like images Contents A29V Index e 2100 expert has improved instrument control Two bioanalyzers can be controlled at one time It is possible to run measurements as well as diagnostics tests on two bioanalyzers at the same time e 2100 expert has improved printing and reporting functions e 2100 expert has extended instrument diagnostics functionality Contents A30V Index Starting 2100 Expert To start 2100 expert
98. d and switched on The Configuration tab now lets you select verification tests to be executed in the verification run New Verification Active Verification All Verifications Configuration Results LogEook B E New Verification E a Installation Verification a gt Software Available Tests o wapc1s46 Apply Name Description Executions Status Select all fa Installation p x Installation Qualification Verifies that files and configurations have been installed to their ap 0 gt Selected _ Hardware Unselect All H System Verification Test Properties Name Installation Qualification Test Description Verifies that files and configurations have been installed to their appropriate location and display correct attributes Approximate Time 300s To select tests check the Apply check box next to the test s Contents A 328 V Index 7 To start the selected tests click Start button in the toolbar The Save As dialog box appears 8 Specify a name and location for the verification results file xvd and click Save The selected tests are executed Running Installation Qualificatior Contents A 329 V Index 9 If atest fails you can Repeat test execution Abort the verification run or skip the current test and Continue with the next test 2100 expert Installation Qualification Test failed Do you want to Re Perform Inst
99. d on and properly connected Check the COM port setting Make sure the bioanalyzer is physically connected to the PC over the serial interface Check the power connection Check the power switch Contents A 181 V7 Index cl If you need additional help please refer to the Agilent 2100 Bioanalyzer Maintenance and Troubleshooting Guide Select an assay for the chip run On the nstrument tab click the Assay button 0R Click the Assays menu Both will open the Assays menu allowing you to select an assay from the submenus Note that you can also select File gt Open File to Run This opens a dialog box allowing you to load either an assay xsy or a chip data file xad The type of assay you have to select depends on the experiment and the staining protocol you use to prepare your cell samples Details on these assays are described in the Application Notes available for each assay Select the desired assay for example Apoptosis The assay is loaded and its name appears on the Information Bar DE1 1700058 APOPTOSIS NOTE After a chip run the results can be evaluated using a different flow cytometric chip data or assay file Refer to Importing Data Analysis Setpoints on page 273 Contents A 182 V Index 6 Select a Destination for the chip data file xad that will be generated as the result of the chip run You can also specify a custom File Prefix for this file r Destination Default C
100. define your own markers and or regions together with gates If you use several markers within one histogram only one of them can be used for gating The other markers can only be used to evaluate regions in the histogram they cover The values belonging to these markers are also displayed in the result table Contents A 213V Index Gating Gating is used to restrict the number of events that are evaluated by gating out filtering events that do not have the fluorescence values set by a marker For example by gating ona blue marker you can exclude all events with low blue fluorescence allowing you for example to gate out dead cells unbound dye and debris Only events with blue fluorescence values within the marker range are evaluated Thereby you can exclude any dead cells and evaluate only the living cells for another property The gating direction defines the reference histogram e Gating from blue to red uses the blue histogram to define the subset by a marker Apoptosis and Antibody Staining assays 2 24h fix Calcein Annexin C 5 No of Evergs No of Events 2 407 10 10 10 108 10 10 10 10 10 10 10t Fluorescence Fluorescence Marker Min Max Ev All Events 0 01 55 43 gt Gated by Calcein 0 22 488 09 gt Calcein on all events 6 88 298 01 Annexin on subset 21 99 3438 99 Contents A 214V Index e Gating from red to blue uses the red h
101. do not use all six wells always load the first sample in well 1 the second sample in well 2 etc Unused wells have to be filled with cell buffer solution otherwise they may run dry during the chip run Because all channels are connected to the priming well this may led to bubbles and to a clogging of the pressure adapter filter to fill both buffer wells with cell buffer Contents A 186 V The cell buffer is used to focus the cells before they pass the detection point You have Essential Measurement Practices Flow Cytometric Assays Handle and store all reagents according to the instructions given in the Reagent Kit Guides Avoid sources of dust or other contaminants Foreign matter in reagents and samples or in the wells of the chip will interfere with assay results Store all reagent and reagent mixes in the dark and refrigerated at 4 C when not in use Allow all reagents to equilibrate to room temperature for 30 minutes before use Protect focusing dye from light The dye decomposes when exposed to light Use appropriate pipette tips For each pipetting step use a fresh new pipette tip Always insert the pipette tip to the bottom of the well when dispensing the liquid Placing the pipette at the edge of the well may lead to poor results due to the formation of a bubble on the bottom of the well i X For chip preparation use inverse pipetting When filling the pipette tip push slightly over the first resistance Empty t
102. e Importing Data on page 271 e Exporting Data on page 277 e Printing Reports on page 286 e Configuring Tables on page 293 Contents A 258 V Index 2100 Expert Data Overview The 2100 expert software manages data in the following different formats e Assay files xsy e Chip data files xad e Comparison files xac e Verification result files xvd e Diagnostics result files xdy e Result flagging rule files xml Assay files Assay files xsy contain the following information e Data acquisition and analysis setpoints Acquisition setpoints are instrument commands and acquisition parameters Analysis setpoints are evaluation parameters some of which you can modify Assay information All parameters defined by the assay such as assay type title and version e Chip and sample information These are chip comments sample names and comments Marker and region definitions flow cytometric assays only Included are associated parameters such as the gating direction Contents A 259 V Index Ladder table and peak table electrophoretic assays only Result flagging rules electrophoretic assays only Chip data files Chip data files xad contain the following information Measurement results After each chip run the measurement results also called raw data are automatically saved in a new chip data file Electrophoretic measurement results are pairs of migration time and
103. e border color of new regions is black To make it easier to differentiate between regions you can change their border color see How to Configure Regions on page 236 Sample 1 10 Region 1 10 10 Tom Toot T 10 Red Fluorescence 3 rrm rrt 10 10 10 10 10 10 Blue Fluorescence Contents A 234V Index To insert an existing region 1 Select the sample where you want to insert an existing region from another sample and click Insert existing region o The nsert Region dialog box appears Insert Existing Region x Existing Regions Region 2 2 Select the region that you want to insert and click nsert Region The region is inserted at its predefined position To remove a region 1 Click the region border to select the region that you want to remove The selected region is highlighted and the Delete Regions button is enabled 2 Click this button The region disappears from the dot plot Contents A 235 V Index How to Configure Regions You can change the color of the region border edit the region s name and define the position and size of the region To configure a region 1 Double click the border of the region that you want to configure 0R Right click the corresponding row in the result table and select Configure Region from the context menu 0R Click the region border to select the region and click the Configure Region button amp in the t
104. e sort the columns by right clicking the table and selecting Columns from the context menu Contents A313 V Index How to Search the Log Book You can search the various log books for any string To search the Log Book 1 cl In the Log Book toolbar click Find ki The Find dialog box appears Enter a search string in the Find What field Use the Column selection list to specify whether you want to search all columns or a particular column only Select the search Direction Find eee Find what instrument Coll Search Soue i st i s sSY Help Cancel Direction Click Find Next If the search string was found in an event the event row gets highlighted in yellow NOTE The search is case sensitive Contents A314 V 6 To continue the search click Find Next Contents A315 V Index Running Instrument Diagnostics 2100 expert provides several tests to check proper functioning of the bioanalyzer hardware You should perform the tests on a regular basis or if incorrect measurements occur You can test the following e Generic bioanalyzer tests which can be run with both types of cartridges electrode or pressure Cartridge e Bioanalyzer in combination with electrode cartridge electrophoresis setup e Bioanalyzer in combination with pressure cartridge flow cytometry setup Contents A 316 V Index Generic Bioanalyzer Tests Diagnostics Test Purpose Electronics Test Fan
105. e values to the X axis The resulting curves show the frequency distribution of the events in relation to their fluorescence intensity values as shown in the following image In real histograms the bin is reduced to a dot data point 50 cells were detected with a fluorescence intensity between 30 and 40 Event Count i 10 3040 100 10 Fluorescence Intensity The histograms can be evaluated statistically with markers that allow you to define ranges of fluorescence intensity values One histogram can be used to represent a range of fluorescence values to define a subset of events Only cells with a fluorescence value within this range are displayed in the second histogram This method is called gating Contents A212 V Index Markers Markers are used to define a range of fluorescence intensity values in a histogram The upper and lower limits of the range are displayed as vertical lines as shown in the following image Lower limit of the marker Upper limit of the marker Event Count Events cells of interest Low intensity Intensity The numerical values for each defined marker are displayed in a separate row in the result table One marker is used as a gate for the second histogram to define a subset of events In predefined assays the markers are set by default and you only need to adjust their position If you want to define your own assays select the Generic assay for acquisition where you can
106. e visible Contents A 230 V Index The content of the result tables depends on the gating direction The histogram that is used for gating can display the following results Marker All events this row shows the data for all measured events for example for all living and dead cells The following rows show the data for the subset of cells defined by the inserted marker If you use a predefined assay the entry can be Calcein on all events for example Min Minimum fluorescence value of the corresponding marker Max Maximum fluorescence value of the corresponding marker Events Number of events covered by the marker For the histogram you use for gating the number of all detected events is displayed in the row All Events total of selected events in relation to the total number of events The row All Events shows 100 of gated of events covered by the marker in the gated histogram Shows no value for the gating histogram Mean Mean fluorescence value of the events inside the marker StdDev Standard deviation to the mean value CV Coefficient of variation GMean Geometric mean Contents A231 V Index The histogram that displays the gated data can show the following data Marker Min Max Events total of gated Mean StdDev CV GMean All events this row shows the data for all events that pass the gate The following rows show the data for all events covered by the i
107. eaks will be assumed to be markers and these may not align with the ladder markers Consequently the software attempts to use the minimum peak height threshold that if it is set low enough will catch the real markers allowing the sample to align NOTE After alignment peaks are shown with relative migration times that are different from the real times with data unaligned Contents A 130 V Index Manual Integration For DNA and Protein assays the 2100 expert software allows to manually integrate peaks Manual integration allows you to move add or delete peak baselines TIP To move a peak baseline point along the vertical line press the CTRL key and left mouse button To move a peak baseline point along the signal press the left mouse button only Example Adjusting peak baselines To manually change peak baselines 1 Switch to the Electropherogram tab in the Data context and zoom into the electropherogram to enlarge the peak of interest 2 Select Electropherogram gt Manual Integration to switch off the automatic integration As an alternative you can click the Manual Integration button U in the toolbar Contents A131 V7 Index The baseline points become visible as blue or green dots Highlighted baseline points are labelled green and can be moved either along the vertical line press CTRL key and left mouse button or along the signal trace left mouse button The blue baseline points are fixed and cannot be moved To high
108. eans the cells are dead If you use a nucleic acid dye you cannot distinguish between living and dead cells you can only count all measured cells See the following image as an example Calcein High fluorescence value indicates living cells Calcein wm D gt Ww ta Low fluorescence value indicates dead or non healthy cells Fluorescence All measured events All events in relation to the blue marker here calcein Living cells related to all measured cells high calcein fluorescence Contents A 24V Index When using the calcein marker in the blue histogram for gating only living cells are considered for building the histogram of the red dye High red fluorescence values indicate living cells with bound antibodies low red fluorescence values living cells without bound antibodies See the following example High fluorescence values indicate binding of labeled antibodies wo t C o gt Ww 4 O zZz Low fluorescence values indicate low binding of labeled antibodies 101 102 108 Fluorescence Percentage of all cells with high red fluorescence selected by the red marker Amount of living cells with high red fluorescence in relation to the amount of living cells Contents A245 V Index Dot plot evaluation If you switch to the Dot Plot tab one region is displayed in the dot plot The red fluorescence values of the region are related to the marker in the
109. ect an assay for the chip run On the nstrument tab click the Assay button 0R Click the Assays menu Both will open the Assays menu allowing you to select an assay from the submenus OR Select File gt Open File to Run This opens a dialog box allowing you to load either an assay xsy or a chip data file xad The type of assay you have to select depends on the required measurement and the Reagent Kit you use to prepare your samples Details on these assays are described in the Application Notes available for each assay and in the Reagent Kit Guide Select the desired assay DNA 1000 for example The assay is loaded and its name appears on the Information Bar DE11 700058 DNA 1000 NOTE After a chip run the results can be evaluated using parameters from a different electrophoretic chip data file xad of the same assay type DNA 1000 in this example Refer to Importing Data Analysis Setpoints on page 273 Contents A52V Index 6 Select a Destination for the chip data file xad that will be generated as the result of the chip run You can also specify a custom File Prefix for this file m Destination Default C DataidsDNADNA 7500 Demo DNA 7500 Custom DataidsDNAIDN4 7500 Demo DNA 7500 A File Prefix 2100 expert max 25 characters Date Acauisition Parameters Run sample gi to fi2 f 7 Under Data Acquisition Parameters enter the number of samples you want
110. ed in the dot plots of all other samples When the properties of the region are changed the changes affect only the selected sample The region is copied to all samples of the assay How to Work with Gates in Dot Plots You can insert gates only in generic assays For predefined assays the gate is already defined Before you can insert a gate you have to draw a region see How to Add Regions to Dot Plots Generic Assay only on page 234 If a gate is already set you first have to remove the existing gate To add a gate to a region 1 Left click the region border to select the region to which you want to add the gate The gating buttons in the toolbar are enabled 2 Click the Horizontal Gate button or the Vertical Gate button F to set a gate along the horizontal or the vertical borders of the selected region In the result table a value appears in the of gated column If the gating direction is already set you first have to remove the existing gate 1 Left click the region border to select the region If a gate already exists the Remove Gate button is enabled 2 Click this button The gate is removed and the gating buttons are enabled Contents A240 V Index Displaying the Results of Regions The measurement results and calculations for regions are displayed in the result table below the dot plot In predefined assays only one region is available while for generic assays dot plots can have as many regions a
111. edures are described in detail in the appropriate Reagent Kit Guide and in the Agilent 2100 Bioanalyzer Maintenance and Troubleshooting Guide Contents A193 V Index Good Practices e Empty and refill the electrode cleaner at regular intervals e g every five assays e The electrode cleaner can be used for 25 assays CAUTION Never use a cloth to clean the electrodes Electrostatic discharge could damage the high voltage power supplies CAUTION Wet electrodes can cause severe damage to the on board high voltage power supplies Always make sure the electrodes are dry before inserting them into the bioanalyzer again Contents A 80 V Index Analyzing and Evaluating the Results of an Electrophoretic Assay The purpose of electrophoretic assays is to separate sample components and determine their size concentration purity or molarity Results for a particular sample are calculated after all data for that sample has been read The steps in data analysis differ depending on the type of assay in use Data Analysis DNA on page 82 Data Analysis RNA and Cy5 Labeled Nucleic Acids on page 87 The RNA Integrity Number RIN on page 90 Data Analysis Protein on page 104 Smear Analysis on page 112 Further steps in analysis are Changing the Data Analysis on page 116 Manual Integration on page 131 Reanalyzing a Chip Data File on page 141 Comparing Samples from Different Electropho
112. en a drop down list Click the sample that you want to use as overlay The histogram curve of the selected sample appears in the histogram view the corresponding entry in the drop down list is marked with a check and a color legend appears above the graph Repeat steps 2 and 3 to overlay further histograms Contents A221 V Index 2 24h fix Annexin C 5 Calcein 4 24h fix 3 24h fix wo g c w gt W pan o z 10 10 Fluorescence To overlay all samples 1 Click the Overlaid Samples 478 button to open a drop down list 2 Select All Samples to overlay the histogram curves of all samples To remove histograms from the overlay 1 Select the sample that contains the overlaid histograms 2 Click the Overlaid Sample button 42 8e to open the drop down list 3 Click the sample that you want to be removed 0R Click No Overlay to remove all overlaid curves from the histogram Contents A 228 V Index How to Set Signal Colors for Overlaid Histograms You can use the Graph Settings tab in the Options dialog box to configure the signal colors colors of curves in histograms 1 Select Tools gt Options 2 Click the Graph Settings tab to bring it to the front xj Data Files Chip Alert Gt i Advanced r Signal Color Signali M Signal P Signal f Signal 2 M Signal M Signal 10 Signl3 signal i Signal 11 sional signals M Signal 12 coat __ To configure t
113. er that is applied to the raw data The setting for the Polynomial Order in the setpoint explorer determines the amount of data to be applied the smaller the number the more data that is applied and the more filtering that takes place Data Points Data points are 0 05 seconds apart Show Data Points is an option that enables the display of the data points used to generate the graph E Electrode Cleaner An electrode cleaner should be used to clean the electrodes after each run is complete The cleaning procedure is slightly different depending upon the type of assay that was just performed DNA or RNA The electrode cleaner looks like a chip except that it is clear With RNA assays you must use two different electrode cleaners one for general cleaning using RNAse free water and another for decontamination using RNAseZAP It is recommended to use a permanent marker to label the electrode cleaners so as not to mix them up Contents A345 V Index Electrokinetic forces Electrokinetic forces are used to move switch and separate the samples Active control over voltage gradients directs the movement of materials using the phenomenon of electrophoretic flow Electroosmotic Flow A phenomenon that results from an electrical double layer formed by ions in the fluid and surface electrical charges immobilized on the capillary walls When an electric field is applied the bulk solution moves towards one of the electrodes This phenomeno
114. erfect RNA sample without any degradation products whereas 1 marks a completely degraded sample The labels in between are used to indicate progressing degradation states of the RNA sample The following figure shows typical representatives for each of the 10 RNA integrity classes Contents A92V Index FU 4 RIN 2 FU 4 3 04 2 54 2 04 1 04 054 0 0 20 25 30 Contents 65 s 20 A93 V Index FU 4 FU 4 FU 4 144 T T 20 25 30 35 40 45 50 55 60 65 s 20 25 30 35 40 45 50 55 60 65 s Contents ADV Index FU 4 FU 4 3S T T 20 25 30 35 40 45 50 5 60 65 s 20 25 30 35 40 45 50 5S 60 65 s Contents A95V Index Computation of the RNA Integrity Number and Signal Anomalies For the computation or the RNA integrity number the electropherogram is partitioned into regions as shown in the figure below The lower marker and the 18S and 28S fragments divide the electropherogram into nine regions 18S a nt Mouse Liver pre region inter region 28S fragment marker precursor region 5S region post region Mouse Liver fast region Signal Anomalies In addition to the computation of the RIN the data analysis detects various unexpected signals disturbing the computation of the RIN Such disturbances are called anomalies Region anomaly detectors recognize unexpected signals
115. eriment microarray real time PCR etc Contents A102 V Index RNA Integrity Number Setpoints Various setpoints are available to customize the display of the RIN RNA Integrity Number With these setpoints you can modify the predefined thresholds for anomaly detection You can find them in the advanced user mode of the setpoint explorer To adjust the setpoints for a single sample switch to the Local tab of the setpoint explorer and open the ANA Integrity Number group To adjust the setpoints for the whole chip switch to the Global tab of the setpoint explorer and open the ANA Integrity Number group in the Sample Setpoints group For the chip you can additionally switch between integer and decimal representation of the RIN For more information on how to use the setpoint explorer see About the Setpoint Explorer on page 117 Contents A 103 V Index Data Analysis Protein The data analysis process for protein assays consists of the following steps 1 Raw data is read and stored by the system for all of the individual samples 2 The data is filtered and the resulting electropherograms of all samples are plotted You can change the settings of the data analysis after the run and reanalyze your data 3 Peaks are identified for all samples and are tabulated by peak ID You can change the settings of the peak find algorithm and reanalyze the data after the run has finished Note that peak find settings can be changed
116. ers It briefly summarizes the necessary steps to prepare and run an assay e Looking at 2100 Expert on page 28 shows how to get started with the 2100 expert software and outlines its main operational possibilities e Running and Evaluating Electrophoretic Assays on page 45 explains how electrophoretic measurements are made using the bioanalyzer gives detailed descriptions of all steps necessary to run electrophoretic assays and shows how to analyze and evaluate results using electropherograms and gel like images e Running and Evaluating Flow Cytometric Assays on page 166 explains how flow cytometric measurements are made using the bioanalyzer gives detailed descriptions of all steps necessary to run flow cytometric assays and shows how to analyze and evaluate results using histograms and dot plots e Working with Chip Data and Assays on page 258 shows you what to do to open save import and export files and how to print the results e Administering System Functions on page 299 is your guideline for configuring the 2100 expert software e Running Instrument Diagnostics on page 316 shows how to use the diagnostic tests to check the bioanalyzer hardware for proper functioning Contents AthV Index e Performing Verifications on page 325 describes how you can validate your bioanalyzer system e Products Spare Parts and Accessories on page 334 lists all parts and accessories includin
117. escence dye 46 G Gates working with 240 Gating 214 direction 225 Gel electrophoresis 352 GFP 168 GFP assays 253 Green fluorescent protein 168 253 Contents H Histogram generating 172 overlaying 227 l Inserting marker 220 Inserting peaks 136 Installation verification 325 Inverse pipetting 187 L Lab on a Chip 350 Laboratories on a microchip 352 Liquid chromatography 352 Log Run log 310 System Log 311 M Manual integration 131 Marker 213 configuring 222 copying 225 inserting 220 limits 224 removing 221 Micro channels 46 Microfluidics 351 Molecular separation techniques 352 A 364 V Index Molecular weight 352 Multi channel mode 73 206 N Navigation 74 207 0 Opening assays 265 Overlay histograms 227 color and scale 229 P Pressure cartridge 189 Products 334 R Raw data 260 Reagent Kit Guides 8 Reagents 186 Region table 114 Regions 175 adding 234 configuring 236 copying 239 size and position 238 Related documentation 8 Removing peaks 134 Result flagging 152 defining rules 159 161 exporting rules 285 Contents importing rules 276 Result table histogram values 230 regions 241 RIN 90 RNA integrity number 90 Run log 310 S Scale overlaid histograms 229 Setpoint explorer 117 Setpoints 262 Show data points 78 210 Sieving polymer 46 Single channel mode 73 206 siRNA Transfection Viability 177 Smear analysis 112
118. estimate the integrity of total RNA samples The RIN extension automatically assigns an integrity number to a eukaryote total RNA sample analyzed on the Agilent 2100 bioanalyzer Using this tool sample integrity is no longer determined by the ratio of the ribosomal bands alone but by the entire electrophoretic trace of the RNA sample including the presence or absence of degradation products In this way interpretation of an electropherogram is facilitated comparison of samples is enabled and repeatability of experiments is ensured Scope What the RIN can do e Obtain an assessment of the integrity of RNA e Directly compare RNA samples e g before and after shipment compare integrity of same tissue across different labs etc Ensure repeatability of experiments e g if RIN shows a given value and is suitable for microarray experiments then the RIN of the same value can a ways be used for microarray experiments given that the same organism tissue extraction method was used What it cannot do e Tell a scientist ahead of time whether an experiment will work or not if no prior verification was done e g RIN of 5 might not work for microarray experiments but might work well for an appropriate RT PCR experiment Also an RIN that might be good for a 3 amplification might not work for a 5 amplification Contents A90 V Index The computation of the RIN is part of data analysis for total RNA samples The computed RNA integrity num
119. ext you can create your own assays based on Agilent templates by modifying certain data for example data analysis setpoints 42100 expert C Eile Context View Assay Properties Windows cS iles a Demo DNA 7500 Custom xs All Samples Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Comparison Sample 11 Sample 12 O Ladder E Instrument m Verification lt lt YS ODDO BROOEI mi System Help E General Properties Assay File Title Location Assay Class Version Modified Comments Copyright 2003 Agilent Technologies 100 bioanalyzer 2100 expert SecuredArea Method Demo Templates dsDNA Demo DNA 7500 Custom xsy Chip Summary Result Flagging Log Books Demo DNA 7500 xsy Demo DNA 7500 C a Method Demo Templates dsDNA DNA 7500 1 3 Mai 19 2005 13 41 40 General Analysis Settings Number of available sample and ladder wells max 13 Minimum visible range s 25 Maximum visible range s 95 Start analysis time range s 26 End analysis time range s 94 Ladder Concentration najul 4 Uses standard area for ladder fragments Lower Marker Concentration ng ul 8 3 Upper Marker Concentration najul 4 2 Used upper marker For quantitation Standard curve fit is Point to Point Show data aligned to lower and upper
120. for all or only certain samples Contents A104 V Index 4 A sizing ladder see the example electropherogram below which is a mixture of proteins of different known sizes is run first from the ladder well The sizes of the individual proteins are preset as kDa in the assay and cannot be changed Please note that the concentrations may vary slightly from ladder lot to ladder lot Size kDa Observations a 6 0 Lower Marker 9 0 Ladder Peak 14 4 Ladder Peak 21 5 Ladder Peak 29 0 Ladder Peak 32 5 Ladder Peak 53 0 Ladder Peak 66 7 Ladder Peak M om Ons UON Peeeerr rs Contents A105 V Index 5 A standard curve of migration time versus size is plotted from the sizing ladder by interpolation between the individual protein size migration points The standard curve derived from the data of the ladder well should resemble the one shown below Point to Point Fit 300 250 200 Size n o 100 50 18 20 22 24 26 28 30 32 34 36 38 40 Time seconds Contents A 106 V Index 6 Two proteins are run with each of the samples bracketing the sizing range The lower marker and upper marker proteins are internal standards used to align the ladder data with data from the sample wells The figure below shows an example of assigned marker peaks in a sample well FU IgG reduced Lower marker 80 Upper marker IgG reduced 15 20 25 30 35 40 m
121. g direction Pan mode or the scales of the X and or Y axes change Scale mode 5 Release the mouse button You can perform several zoom pan and scale steps in a row To undo the last zoom pan or scale step 1 Click the Undo Zoom button or double click in the electropherogram To undo all zoom pan and scale steps 1 Click the Undo All button Contents A7l1lV Index To display data points in an electropherogram 1 From the Electropherogram menu select Show Data Points or click the button in the toolbar Data points used to generate the graph are now shown as bullets Data points are 0 05 seconds apart To remove the gray to white gradient from the background of an electropherogram 1 From the Electropherogram menu select Show Gradient The color gradient disappears and a white background is displayed To show hide the grid lines on an electropherogram 1 From the Electropherogram menu select Show Grid Contents A7l8V Index Cleaning the Electrodes after an Electrophoretic Assay When the assay is complete remove the used chip from the bioanalyzer and dispose of it according to the guidelines established by your laboratory safety officer Remove the chip quickly to prevent a buildup of residues from the solutions on the electrodes Electrodes Then perform the cleaning procedure to ensure that the electrodes are clean i e no residues left from the previous assay The cleaning proc
122. g reorder numbers that are required for electrophoretic and flow cytometric measurements e Glossary on page 338 explains terms in context with flow cytometry electrophoresis and terms specific to the bioanalyzer software and hardware If you have any questions this manual cannot answer please refer to the supplemental literature listed in Related Documents on page 8 If you still have questions contact Agilent for additional support at http www agilent com chem labonachip Contents AlV Index Related Documents A collection of supplemental literature is given in the following Bioanalyzer Manuals Publication Number Title G2938 90006 Agilent 2100 Bioanalyzer Installation and Safety Manual G2946 90003 Agilent 2100 Bioanalyzer Maintenance and Troubleshooting Guide CD ROM Publication Number Title G2946 60002 Agilent 2100 Bioanalyzer How to Use Multimedia CD ROM Reagent Kit Guides The Reagent Kit Guides give you information on how to perform specific assays including sample and chip preparation Publication Number Title G2938 90300 Kit Guide Binder English including all Reagent Kit Guides G2938 90010 Reagent Kit Guide DNA 500 and DNA 1000 Assay G2938 90020 Reagent Kit Guide DNA 7500 and DNA 12000 Assay Contents A8V Index Publication Number Title G2938 90030 Reagent Kit Guide RNA 6000 Nano Assay G2938 90040 Reagent Kit Guide RNA 6000 Pico Assay G2938 90050 Rea
123. ge gradient similar to slab gel electrophoresis Because of a constant mass to charge ratio and the presence of a sieving polymer matrix the molecules are separated by size Smaller fragments are migrating faster than larger ones Dye molecules intercalate into DNA or RNA strands or protein LDS micells These complexes are detected by laser induced fluorescence Data is translated into gel like images bands and electropherograms peaks With the help of a ladder that contains components of known sizes a standard curve of migration time versus fragments size is plotted From the migration times measured for each fragment in the sample the size is calculated Two markers for RNA only one marker are run with each of the samples bracketing the overall sizing range The lower and upper markers are internal standards used to align the ladder data with data from the sample wells This is necessary to compensate for drift effects that may occur during the course of a chip run For DNA and protein assays quantitation is done with the help of the upper marker The area under the upper marker peak is compared with the sample peak areas Because the concentration of the upper marker is known the concentration for each sample can be calculated Besides this relative quantitation an absolute quantitation is available for protein assays using external standard proteins For RNA assays quantitation is done with the help of the ladder area The
124. ged Contents A120 V Index Filtering Setpoints The first step the software takes in analyzing the raw data is to apply data filtering The following filtering setpoints can be changed Filter Width Defines the data window given in seconds used for averaging The broader the filter width the more raw data points are used for averaging As a result the noise level will decrease but peaks will become lower and broader Overall changing the Filter Width has more effect on the result of the filtering procedure applied then does changing the Polynomial Order Polynomial Order This setting is used to define the power series applied to fit the raw data The higher the number the more the fit function will follow the noisy raw data curve As a result the noise level of the filtered curve will increase Integrator Setpoints After data filtering the peak find algorithm locates the peaks and calculates the local peak baselines The algorithm begins by finding all the peaks above the noise threshold in order to determine the baseline after which any peaks below the noise threshold are rejected A local baseline is calculated for each peak to allow for baseline drift Contents A121 V7 Index The four integrator setpoints that can be changed are Slope Threshold The Slope Threshold setpoint determines the difference in the slope that must occur in order for a peak to begin The inverse of this value is used to determine the peak end
125. gent Kit Guide Protein 200 Plus Assay G2938 90060 Reagent Kit Guide Protein 50 Assay G2938 90070 Reagent Kit Guide Cell Fluorescence Assays G2938 90080 Reagent Kit Guide Cell Fluorescence Checkout Kit Application Notes and Technical Notes Application Notes and Technical Notes are available from the Agilent 2100 Bioanalyzer Help Desk or from the lab on a chip web pages http www agilent com chem labonachip Newly Published Documentation Follow this link to see if there is any new documentation http www chem agilent com scripts Library asp Contents ASV Index How to Use this Manual This manual uses convenient online navigation features and follows certain typographic conventions Online Navigation Use the interactive bookmarks in this frame to move to your desired topic Use Acrobat Reader s navigation bar to move around within a topic Bi Adobe Acrobat eBook pdf bn File Edit Document Tools View Window Help T amp Bookmark x L Agilent 2100 Bioanalyzer 1 2100 expert User s Guide H About this Manual Quick Start The following step by step instructions summarize the basic steps needed to perform a measurement with the Agilent 2100 bioanalyzer L Operating Basics L Switching Between E L Running and Evaluatit C Running and Evaluatir L Configuring 2100 exp L Running Instrument C L Performing Qualificati H 2100 expert
126. gmentsize Concentration Molarity Observations 1 15 00 4 20 424 24 CalculatedLowerMarker 2 472 30 0 76 2 45 3 13 40 500 66 0 34 1 03 4 84 13 1500 00 2 10 2 12 CalculatedUpperMarker The following example demonstrates how to add the migration time to the Peak Table Contents A 293 V Index Showing and Hiding Columns To add the Aligned Migration Time column to the table 1 Right click the heading row of a table and select Configure Columns from the context menu The Configure Columns dialog box opens eer Configure Columns X E Configure Columns po Oooo E3 Copy To Clipboard trl C 1500 00 weer Marker Contents A294 V Index 2 Move any desired column headers from the Available list to the Displayed list xi Available Displayed Observations Up Down Cancel Reset Help 3 Configure the order of the column headers in the Displayed list by using the Up and Down buttons 4 Click OK A new column Aligned Migration Time is inserted in the table Size bp Conc ngul Molarity nmol l Aligned Migration Time s Lower Marker Contents A295 V Index Changing the Column Sequence TIP You can set the column sequence also using the Up and Down buttons in the Configure Columns dialog box To change the column sequence of a table 1 Position the mouse pointer on a column header 2 Click and hold the left mouse button and dr
127. gramme Agilent 2100 bioanalyzer 2100 expert reports Passed C Programme Agilent 2100 bioanalyzer 2100 expert assays Passed C Programme Agilent 2100 bioanalyzer 2100 expert data packets Passed C Programme Agilent 2100 bioanalyzer 2100 expert assays demo flow cytometry 14You can now navigate to other test categories and execute additional verification tests Contents A 332 V Index 15When you close the verification result file File gt Close try to switch to another context or exit 2100 expert the following message appears 2100 expert xi Validation File will no longer be active All results upto the last run will be saved as a read only file Do you want to continue If you select No you return to the Verification context and can run further verification tests If you select Yes the verification result file xvd is closed and becomes read only NOTE You can re open verification result files only for viewing and printing TIP Select File gt Print to generate a printed report of the verification run Contents A 333 V Index Products Spare Parts and Accessories To buy the following products spare parts and accessories for the Agilent 2100 bioanalyzer please refer to the Agilent Online Store http www agilent com home buyonline html Bundles G2940CA Agilent 2100 bioanalyzer desktop system Includes Agilent 2100 bioanalyzer HP Compaq desktop PC color printer system software vortexer and acce
128. grammeAgilent 2100 bioanalyzer 2100 expert Data Method Browse Reset Data File Format Binary Format KL Format X Mr Admin as 2100 Administrator Auto Export Auto Print Contents A42V Index Closing 2100 Expert To close the 2100 expert software 1 From the File menu select Exit If a chip run is in progress the following message appears 2100 expert x oe l Run is being performed in the Instrument Context Cannot terminate application Click OK and wait until the chip run is complete If there are unsaved files open the following dialog box appears 2100 expert x Save changes to the following files Contents A43 V Index NOTE This dialog box may also appear if you try to switch between contexts while there is unsaved data 2 Click Yes to save the changes to the selected files and continue quitting 2100 expert If you want to save changes only to particular files select these files in the list by single clicking them By default all files with unsaved changes are selected If you click No 2100 expert quits without saving any changes If you do not want to quit 2100 expert at this time click Cance to return to your 2100 expert session without saving anything After you have confirmed the messages 2100 expert quits Contents AMV Index Running and Evaluating Electrophoretic Assays For running and evaluating electrophoretic assays you
129. h assay Staining Cells on page 168 explains the principle e Next the stained cells are analyzed on the chip They pass the detector in single file and are analyzed individually for their red and blue fluorescence intensities The results are displayed as histograms or dot plots Refer to Cell Detection with the Agilent 2100 Bioanalyzer on page 170 for a detailed explanation Contents A 167 V Index Staining Cells With the 2100 expert software you can differentiate several properties of a cell The characteristics that are examined depend on the dye which binds specifically to a cellular constituent or is metabolized by the cell to generate a fluorescent product You usually use two dyes with different colors Typically one of the two dyes Is used as reference dye to select the target cells living dead cell line type etc The second dye can be used to detect another characteristic of the cell Recommended dyes The tables below list dyes that match to the detection optics specification Excitation max 470 amp 630 nm Emission max 525 amp 680 nm The following dyes are recommended for use as the blue stain Dye blue fluorescence Max Excitation Max Emission wavelength wavelength Calcein living cell stain 493 nm 514 nm Cell Tracker green cell tracing 492 nm 517 nm viability stain GFP green fluorescent protein 490 nm 510 nm SYT016 DNA dye 485 nm 530 nm Contents A 168 V Index The fo
130. he pipette tip only to the first resistance This procedure avoids the introduction of bubbles and ensures pipetting the right volume Never leave any wells empty or the pressure adapter may become clogged Pipette 10 ul of cell buffer or sample replicate in any empty sample well Contents A 187 V Index e Before bead preparation vortex bead vials for 15 seconds e Prepared chips must be used within 5 minutes If a chip is not run within 5 minutes beads may settle or reagents may evaporate leading to poor results e Never touch the instrument lens e Never touch the Agilent 2100 bioanalyzer during a chip run and never place it on a vibrating ground Contents A 188 V Index Loading the Cell Chip into the Bioanalyzer The Agilent 2100 bioanalyzer uses different cartridges for electrophoretic and flow cytometric assays For flow cytometric measurements the pressure cartridge is required The pressure cartridge contains a tubing and filter assembly that connect to the vacuum pump The seal has to match the priming well on the chip so that the required low pressure can be built up The pressure cartridge can be identified by the engraved number 2 on the front NOTE Any cartridges without engraved numbers are electrode cartridges Contents A 189 V Index If the bioanalyzer is set up for electrophoretic assays but you want to run flow cytometric assays proceed as follows 1 Open the lid and pull down the metal locki
131. he signal color 1 Click the colored square corresponding to the signal The Color dialog box appears 2 Select a color for the signal and assign it by clicking OK Contents A229 V Index Displaying the Results of Histogram Evaluations The calculated results are displayed in result tables one table below each histogram Markers gates several statistical values and the values of events are shown in the result tables Marker Min Max Events Total 1 of gated Mean StdDev CY GMean gt All Events 0 02 51 42 1606 7 13 88 37 4 51 Gated by Calcein 0 04 51 42 1418 ee 86 47 GUE CD3 APC on all events 1 18 4085 88 1356 CD3 APC on subset 1 18 4085 88 1195 Each marker you insert in the histogram gets its own row Note that you can only use one marker for gating The additional markers can be used to evaluate different parts of the histogram statistically If the option Hide superset curve is disabled in the setpoint explorer two additional rows are displayed in the gated histogram s result table The superset curve shows a histogram of all measured events the gate is not considered You can export the table data for further evaluation in other applications See Exporting Data on page 277 NOTE The layout of the result table can be configured see Configuring Tables on page 293 Not all of the values listed below may therefore b
132. ics Test Tests instrument electronics of Executed passed x Fan Test Tests if instrument fan is working of Executed passed x Lid Sensor Test Tests if the lid sensors are working of Executed passed gt x Temperature Test Checks if the temperature sensors and heater a Executing x Stepper Motor Test Tests if horizontal and vertical motors are workin SyExecution pending New x Electrode Diode Test Tests conductivity of channels pin to pin Execution pending fat Xx High Voltage Stability Test Tests high voltage accuracy and stability Execution pending El wl UU Arm wams Chaek af bha hinh sinlbana anbeallas Ra Cuan binan nandina Test Properties Bioanalyzer hardware test x Please insert Cartridge and an empty chip Close the lid and press OK ID 4 Name Temperature Test Description Checks if the temperature sensor Approximate Time 360s Limits Heater test destination Temperature 40 C Heater Ramp Timeout 300 s Duration of stability test 45 s Required Temperature Stability 0 4 C Requirements Configuration All selected tests are performed Contents A 323 V Index The Status column indicates the status of each test Executing Execution pending Executed passed Executed failed 7 If any test failed redo the test 8 If failures still persist contact Agilent service The results of diagnostics tests are stored in xd
133. ilent 2100 bioanalyzer 2100 3 Security Service System Apr 26 2005 07 53 50 expert Secured4reaMethod Demo Templates dsDNA Demo DNA 7500 xsy 2100 Administrator as 2100 Administrator has started 2100 expert Version B 02 01 51209 0 User Interface Apr 26 2005 07 53 18 File has been modified C Programme Agilents2100 bioanalyzers2100 3 Security Service System Apr26 2005 07 53 18 expert Secured4reaS ystem SystemFileLogs xml e File has been modified C Programme Agilent 2100 bioanalyzer 2100 3 Security Service System Apr 26 2005 07 53 17 expert Secured4reaSystem SystemFileLogs xml 2100 expert security service started 0 Security Service System Apr 26 2005 06 34 16 O ES Security Guard has been started to watch C Programme Agilent 21 00 bioanalyzer2100 0 Security Service System Apr 26 2005 06 34 16 7 experthSecured amp rea 2100 expert ended Version B 02 01 51209 0 User Interface Apr25 2005 12 45 45 2100 Administrator as 2100 Administrator has succesfully unlocked the application 0 User Interface Apr 25 2005 12 45 40 2100 Administrator as 2100 Administrator has started 2100 expert Version B 02 01 51209 0 User Interface Apr 25 2005 11 02 26 X wooo e Index The system log book is saved in config SystemFile xml The log book entries can be exported from this file How to Change the Display of the Log Books To sort a log book table 1 Click the column header you want to sort the table by The log book table is sorted by the en
134. in each region If detected the anomaly is displayed in the Error sub tab of the Electropherogram and Gel tab Contents A 96 V Index Anomaly Description Critical Unexpected baseline signal Yes Unexpected signal in pre region No Unexpected signal in 5S region Yes Unexpected signal in fast region Yes Unexpected signal in inter region Yes Unexpected signal in precursor region No Unexpected signal in post region No Unexpected ribosomal ratio Yes Unexpected sample type Yes Unexpected lower marker compared to previous well No Two categories of anomalies were introduced critical and non critical Anomalies in regions interfering with the customer sample RNA are considered critical The corresponding gel lane is flagged red The baseline anomaly for example is detected for signals with fluctuating or steep baseline The ribosomal ratio anomaly detects unexpected ratios of the 28S fragment area and the 18S fragment area The unexpected sample type anomaly is detected for samples which do not fit the standard total RNA profile Contents A9IITV Index If a non critical anomaly is detected the RIN can still be computed accurately Therefore non critical anomalies are not flagged Non critical region anomalies are pre region anomaly precursor region anomaly and post region anomaly The electropherogram below gives an example for a non critical anomaly in the post region Samale 3 anomaly in the post region Contents A
135. ir of data points that are used to interpolate data between those points Contents A 354 V Index Polynomial Filter The first step 2700 expert takes in analyzing the raw data is to apply data filtering Data filtering is done by means of a polynomial filter that is applied to the raw data Priming Station Consists of a chip holder that has a syringe mounted on the lid that seals over the chip The syringe is used to force the buffer solution loaded into the well marked G with a circle around it into all the passageways inside the chip prior to running it in the bioanalyzer Contents A 355 V Index S Serial port The serial ports COM ports are used to connect your computer with the Agilent 2100 bioanalyzer The number of available ports depends on the computer you use Signature Signatures are available in the 2100 expert software only with the security pack installed All activities on data such as creating modifying and deleting data must be confirmed by the user with an electronic signature user name and password By requesting this signature it is ensured that only authorized users can create modify and delete data Slope Threshold The Slope Threshold setpoint determines the difference in the slope that must occur in order for a peak to begin The inverse of this value is used to determine the peak end Standard Curve The standard curve is obtained by plotting the size of the ladder peaks vs time using a p
136. istogram to define the subset by a marker GFP assay Sample 6 GFP No of Events 5 8 6 4 2 D 4 4 107 1407 10 10 10 10 10 10 10 10 Fluorescence Fluorescence Marker Min Max Event Marker Min Max Ev b Gated by CBNF 0 01 272 99 E All Events 0 02 409 07 GFP onsubset 1 26 851 03 z p CBNF onal events 1 70 2869 80 NOTE Predefined assays have a fixed gating direction while assays of type Generic have a variable gating direction Contents A 215 V Index The following figures illustrate gating from blue to red The two histograms display all measured events in the blue histogram and in the red histogram without gating In this case you cannot see which cells fluoresce only in the blue and which fluoresce only in the red A 3 5 38 o All events that show red fluorescence gt gt Ww Ww All events that show blue fluorescence Intensity Intensity Contents A216 V Index By setting a marker on the blue histogram you can define the blue fluorescence range that must be met for a cell to be considered for the red histogram You use the gating on the blue histogram to define a subset for the red histogram A A g z 8 8 Subset of the events S 5 defined by the marker i i n fie Events of the subset that n show also red fluorescence intensity Intensity gt Contents A 217 V Index The red histogram
137. ith the pressure cartridge G2938 68100 Test Chip Kit for Electrophoretic Assays Comprises 1 Autofocus 1 Electrode Diode and 5 Leak Current Clips G2938 68200 Test Chip Kit for Flow Cytometric Assays Comprises 1 Cell Autofocus Chip G2938 81605 RS 232 cable Communication cable PC Agilent 2100 Bioanalyzer 2110 0007 Fuse 5042 1398 Adjustable Clip for use as spare part for the chip priming station 5065 4401 Chip Priming Station including gasket kit and adjustable clip G2938 68716 Gasket Kit Includes spare parts for the chip priming station 1 plastic adapter 1 ring and 10 gaskets 5065 4428 IKA Vortexer 115V Must be ordered at IKA http www ika de 5065 4429 IKA Vortexer 230V Must be ordered at IKA http www ika de Contents A 336 V Index e 5065 9966 Vortex Mixer Adapter for IKA vortexer e 5065 9951 Electrode Cleaner Box Contains 7 electrode cleaners e G2946 60002 Agilent 2100 bioanalyzer How to Use CD ROM Contains videos showing the chip preparation for all assays and the hardware maintenance Contents A 337 V Index Glossary This glossary explains terms in context with flow cytometry electrophoresis and software or hardware of the Agilent 2100 bioanalyzer A Area Threshold The Area Threshold setpoint determines the minimum amount of peak area that must be detected before a peak is recognized Assay An assay is a solution with defined chip chemicals instr
138. l graphic files This applies to all graphs that can be displayed in 2100 expert such as electropherograms or dot plots To export a graph 1 Right click the graph and select the appropriate entry e g Save Gel or Save Electropherogram from the context menu 0R Click the button in the toolbar The Save As dialog box appears 2 Under File name enter a name and choose the destination directory 3 Under Save as type select a graphic file format bmp jpg wmf tif or gif 4 Click Save The graph is written to the specified file Contents A 282 V Index Note the following e Histograms only one histogram graph is exported either the red or the blue histogram e Electropherograms if the grid view is active an overview image of the electropherograms of a samples and the ladder is exported TIP Electropherograms gel like images histograms and dot plots can be automatically exported every time a chip run has finished Refer to Exporting Chip Run Data Automatically on page 280 for details Copying Graphs and Tables into the Clipboard You can copy graphs into the clipboard This applies to all graphs that can be displayed in 2100 expert such as electropherograms or dot plots You can also copy tables or parts of tables into the clipboard This applies to most of the tables that can be displayed in 2100 expert such as result tables or log book tables To copy a graph or table into the clipboard 1
139. le the gel dye mix Load the gel dye mix using the priming station Load the DNA RNA marker solution and buffer Load the destaining solution for protein assays Load the chip with ladder and samples Contents Ab4V Index Essential Measurement Practices Electrophoretic Assays General WARNING Wear hand and eye protection and follow good laboratory practices when preparing and handling reagents and samples WARNING No data is available addressing the mutagenicity or toxicity of the dye DMSO reagent Because the dye binds to nucleic acids it should be treated as a potential mutagen and used with appropriate care The DMSO mixtures should be handled with particular caution as DMSO is known to facilitate the entry of organic molecules into tissues We strongly recommend using double gloves when handling DMSO mixtures e Handle and store all reagents according to the instructions given in the Reagent Kit Guides e Avoid sources of dust or other contaminants Foreign matter in reagents and samples or in the wells of the chip will interfere with assay results e Always insert the pipette tip to the bottom of the well when dispensing the liquid Placing the pipette at the edge of the well may lead to poor results due to the formation of a bubble on the bottom of the well Contents A55V Index i X Protect dye and gel dye mix from light Remove light covers only when pipetting Dye decomposes when exposed to light Use a new
140. lectropherogram IgG non reduced All Comparison Files Comparison Summary Gel Electropherogram K ComparisonFile0 Protei oO IgG non reduced C IgG nonreduced O Ladder C IgG reduced O Sample 1 IgG non reduced 2 a S F 5 a S g S iewport 1 IgG non reduced Ladder Legend Errors Contents A149 V Index 9 Select the Ge tab to display a comparison of the gel like images of the samples Gel IgG non reduced All Comparison Files E ComparisonFile0 Protei C IgG non reduced IgG nonr Ladder Ladder IgG redu IgG redu Sample 1 C IgG nonreduced C Ladder 5 IgG reduced C Sample 1 0 0 Lower Marker e __ 8 ___o _bet_aapsten ea J sa 26 8 oof 100o a b zooj tooo 0 0 Upper Marker 4 Results Peak Table Note that the Gel Tab has the same functionality as in the Data context Contents A 150 V Index 10 From the File menu select Save to save the comparison file xac under the default name or select Save As to save it under a new name The default name is derived from the assay class ComparisonFileX Assay Class xac where X is an autoincremented number Example ComparisonFile0 Protein 200 xac NOTE You can re open comparison files to review the comparison results and to add remove samples Contents A151 V7 Index Result Flagging Result flagging can be used to assign a user defined color code
141. lectrophoretic assays only e Define markers and regions for evaluation flow cytometric assay Generic only For example if you want to adjust marker positions and use these for future chip runs You can modify custom assays at any time See How to Modify a Custom Assay on page 265 Contents A 262 V Index TIP If you just want to view the properties of a custom assay you can open the assay file in read only mode ensuring you do not make accidental changes The Assays menu is dynamically built from the structure and contents of the assays subdirectory of the 2100 expert installation folder C Program Files Agilent 2100 Bioanalyzer2100Xpert assays electrophoresis protein Sel x File Edit view Favorites T Help Oba O F X GP search Folders fe G X I Te Address C Program Files Agilent 2100 eee ai gt gt Go Folders _Size Type Date Modified 5 Aglent rc eme Protein 50 xsy 50KB XSYFile 7 14 2003 2 54 PM 2100 Bioanalyzer Err reece SOKB XSY File 7 14 2003 2 54 PM B 2100Xpert assays EMS demo electrophoresis i flow cytometry a electrophoresis o dsDNA other protein O RNA i flow cytometry bin xl Type XSY File Date Modified 7 14 2003 2 54 PM Size 49 0 KB 49 0 KB My Computer TIP You can add items to the Assays menu by placing assay xsy files yo
142. light a baseline point click it Sample 3 TIP If you want to change several baseline points deactivate the automatic analysis by clicking the Pause Analysis button in the toolbar This way the software will not recalculate the data analysis with every change Once you have changed all baseline points click the Pause Analysis button again to activate automatic analysis Contents A 132 V Index 3 Adjust the baseline points as appropriate TIP To move a peak baseline point along the vertical line press the CTRL key and the left mouse button To move a peak baseline point along the signal press the left mouse button only 4 Click the Automatic Analysis button SI to enable the integration again The integration results in the result and peak tables will change according to the changes done Contents A 133 V Index Example Removing peaks To remove peaks 1 Highlight the E ectropherogram tab in the Data context and zoom into the electropherogram to enlarge the peak of interest 2 Select Electropherogram gt Manual Integration to switch off the automatic integration As an alternative you might click the Manual Integration button UF in the toolbar The baseline points become visible as blue or green dots JP undo Zoom Copy Electropherogram ta Save Electropherogram Size kDa Rel Conc ug ml Calib Conc ug ml Total Observati 1 4 6 0 0 0 0 0 0 0 Lower Marker A
143. like images to make details better visible See How to Change the Display of Electropherograms and Gel like Images on page 75 Contents Al2V Index How to Switch Between Single View and Grid View Electropherograms To switch between single view and grid view 1 From the Electropherogram menu select View Single Sample or View All Samples 0R Click the View Single Sample or View All Samples button on the Electropherogram toolbar 0R Click the A Samples entry in the Tree View Panel to switch to the grid view or any sample name to switch to the single view 0R Double click any electropherogram the grid view to switch to single view PCR Mix 2 FU 100 80 60 40 20 PCR Mix 2 PER A PCR d d d o b h f a I a EEEE tt j 4 a t an oo gt 2 Size bp Conc ng ul Molarity nmol l Observations al 4 15 4 20 424 2 Lower Marker zm 151 2 35 23 6 3 159 3 83 36 6 4 197 3 78 29 0 E 206 4 82 35 4 6 293 3 42 17 7 xl Results Peak Table Legend Errors Contents Size bp Conc na ul Molarity nmol l Observations d 15 4 20 424 2 Lower Marker 2 151 2 35 23 6 3 159 3 83 36 6 4 197 3 78 29 0 E 206 4 82 35 4 6 293 3 42 17 7 X Results Peak Table Legend Er
144. ll should resemble the one shown below Point to Point Fit 800 700 600 500 400 300 200 100 0 40 45 50 55 60 65 70 75 80 85 90 95 100 105 Time seconds Size Contents AS4V Index 6 Two DNA fragments are run with each of the samples bracketing the DNA sizing range The lower marker and upper marker are internal standards used to align the ladder data with data from the sample wells The figure below shows an example of assigned marker peaks in a sample well FU Ladder Lower marker Upper marker 40 ip 30 3 20 0 30 40 50 60 70 80 90 100 110 s NOTE The software performs alignment by default Turning automatic data analysis off suspends data analysis until you turn it on again 7 The standard curve in conjunction with the markers is used to calculate DNA fragment sizes for each sample from the migration times measured Contents A85V Index 8 To calculate the concentration of the individual DNA fragments in all sample wells the upper marker in conjunction with an assay specific concentration against base pair size calibration curve is applied to the individual sample peaks in all sample wells NOTE The software allows you to redefine the peaks chosen as upper and lower markers A change in marker selection will cause quantitative changes in the calibration procedure however and therefore in the entire data evaluation 9 If the check box Rest Digest on the Chip Summary
145. llowing dyes are recommended for use as the red stain Dye red fluorescence Max Excitation Max Emission wavelength wavelength CBNF Carboxynaphthofluorescein 595 nm 675 nm living cell stain APC Allophycocyanin intra and 650 nm 660 nm extra cellular antibody staining Cy5 labeled Streptavidin and 647 nm 665 nm labeled anti IgG Apoptosis intra and extra cellular antibody staining Alexa 647 650 nm 668 nm Contents A 169 V Index Cell Detection with the Agilent 2100 Bioanalyzer LabChip technology allows cell measurements by integrating cell flow hydrodynamic focusing and fluorescence detection into a microfluidic chip A cell suspension can be confined or pinched to a portion of a microfluidic channel causing cells to line up in single file for individual cell detection The following images illustrate the pinching process Buffer well Sample well Pinching area Cells Detector Cell Buffer Up to six cell samples can be analyzed on a chip They are measured sequentially Contents A170 V Index Measuring Events The bioanalyzer counts cells stained with fluorescent dyes and measures their fluorescence intensities Each cell or bead that passes the detector and emits fluorescence above a threshold value is counted as an event For each event the intensity of two different fluorescent signals red and blue is recorded The intensity of the fluorescent signal depends on the
146. ls Once inside the cells the colorless AM ester is cleaved by esterases resulting in the formation of the highly fluorescent calcein The number of events resulting from a calcein related staining thus gives you the Contents A241 W Index number of living cells in a sample For detailed information refer to the application note Apoptosis Detection by Annexin V and Active Caspase 3 with the Agilent 2100 Bioanalyzer Apoptotic cells In apoptotic cells phosphatidylserine is no longer confined to the inner leaflet of the plasma membrane bilayer Phosphatidylserine becomes accessible on the outer surface of the cell membrane and can be bound with high affinity by the protein annexin V which can be labeled with biotin or dyes such as Cyb Gating direction The gating direction is from blue fluorescence living cells to red fluorescence annexin Histogram evaluation The two histograms displaying the results of the assay are related to calcein blue fluorescence and annexin V red fluorescence High fluorescence values in the blue histogram indicate living cells low values correspond to dead cells See the following image as an example Contents A248 V Index Calcein High fluorescence value indicates living cells alcein D i o gt Ww a E z Low fluorescence value indicates dead cells The values are displayed in the result table each histogram has its own table All measured events
147. lts e Exporting Tables on page 281 e Exporting Graphs on page 282 e Copying Graphs and Tables into the Clipboard on page 283 Information that you have entered to document a chip run can be exported for reuse in future chip runs e Exporting Chip Sample and Study Information on page 284 From electrophoretic assay or chip data files you can also export rule definitions for result flagging e Exporting Result Flagging Rules on page 285 Contents A 271 W Index Exporting Chip Run Data To export chip run data 1 Switch to the Data context 2 Inthe Tree View Panel select a chip data xad file or load a file 3 From the File menu select Export If you selected an electrophoretic chip data file the Electrophoresis Export Options dialog box appears If you selected a flow cytometric chip data file the Flow Cytometry Export Options dialog box appears Flow Cytometry Export Options Flow Cytometry Export Result Tables Create a csv file containing result table values IV Sample Data Export textual list files one file per sample I FCS Data Create FCS standard files one file per sample IV Dot Plot Images x Formats Export the Dot Plot image one file per sample Histogram Images Formats Export the blue and red histogram images one file per sample Export to XML Export the complete data file into self describing XML format Export Directory Defa
148. lues Contents A 155 V Index A result flagging rule consists of the following e Label Expression An optional description for the rule used to label samples meeting this rule If Expression An expression built from predefined functions variables and logical operators e Comment An optional comment for the rule Color Expression A solid color or a color gradient built from two colors used for flagging samples that meet the rule How to Use the Form Mode on page 159 shows how to proceed when defining rules You can reuse result flagging rule definitions see Exporting Result Flagging Rules on page 285 and Importing Result Flagging Rules on page 276 Contents A 156 V Index Color Indication The results of the result flagging rules is displayed e On the Chip Summary Tab General Properties Assay Properties Chip Summary Gel Electropherogram l Result Flagging l Log Book l A z 3 2 2 z DNA LabChip Siting A Data File Location Created Modified Software ae File Version 2100 expert_DNA 7500_00000_2005 04 27_11 34 48 xad C alyzer 2100 expert Secured4rea Data dsDNA DNA 7500 Demo DNA 7500 April 27 2005 11 34 55 April 27 2005 12 54 40 Created by version B 02 01 51209 modified by B 02 01 51209 8 Latest version prepared by laura Chip Comments N
149. ml files To export result flagging rules 1 Open the electrophoretic assay or chip data file with the desired result flagging rules in the respective context 2 Switch to the Result Flagging Tab 3 In the Result Flagging toolbar click S The Save Rule Definitions dialog box appears 4 Browse for a folder where you want to store the rules and specify a name for the xml file 5 Click Save Contents A 285 V Index Printing Reports For documentation and presentation purposes you can print reports for assay xsy chip data xad verification results xvd and comparison xac files You can print all reports manually see How to Print a Chip Run Report on page 287 When printing manually a preview function allows you to view the printout before starting the print job The 2100 expert program can also be set to print customized chip run reports automatically at the end of the run These reports can be set up to contain different information settings for the manual and automatic print functions are maintained separately See How to Turn on and Configure Automatic Printing of Chip Run Reports on page 291 for more information TIP Beside sending reports to a printer you can also create pdf and html files Contents A 286 V Index How to Print a Chip Run Report The following information can be included in a chip run report You can always include Run summary general data about the assay and s
150. mport Sample Information dialog box appears 3 Select the file that contains the information you want to import and click Open The Sample Information and Study Information sub tabs update to show the imported data 4 From the File menu select Save to make the changes permanent Contents A 275 V Index Importing Result Flagging Rules You can import result flagging rules into electrophoretic assay xsy or chip data xad files Result flagging rules can be stored in xml files see Exporting Result Flagging Rules on page 285 To import result flagging rules 1 Open an electrophoretic assay or chip data file in the respective context 2 Switch to the Result Flagging Tab 3 In the Result Flagging toolbar click g The Load Rules dialog box appears 4 Select the xml file that contains the set of result flagging rules and click Open The imported rules appear in the rule list Contents A 276 V Index Exporting Data 2100 expert allows you to export the results of your chip runs in a variety of formats The exported data can be used for further evaluation with other applications such as text processors graphic tools MS Excel or flow cytometry applications You can export the chip run data of the currently loaded file either manually or automatically e Exporting Chip Run Data on page 278 e Exporting Chip Run Data Automatically on page 280 If you want to export only parts of your measurement resu
151. n additional software license 1 Select Registration from the Help menu to open the License Administration Tool window License Administration Tool x j Add Licence Select Product Agilent 2100 Bioanalyeer x 2100 Bioanalyzer he Module licensekey Added 2 Switch to the Add License tab 3 In the Select Product field the Agilent 2100 Bioanalyzer must be selected Contents A 307 V Index 4 Inthe Select Module field select the license for the software module that you want to activate xi View License Add Licence Select Product Agilent 2100 Bioanalyzer Select Module 2100 electrophoresis license s 2100 electrophoresis license 2100 flow cytometry license 2100 security pack license License Key 5 Enter the correct License Key and click the Add button A message box informs you whether the license key was added successfully 6 Ifyou want to add more licenses repeat the previous two steps for every license key 7 Click the Exit button to close the License Administration Tool window The licensed software modules are now activated and can be used NOTE If you added the license key to activate the security pack the 2100 expert software closes and the secured file area will be set up Follow the instructions displayed in the different pages of the setup wizard Contents A 308 V Index NOTE Store your license keys in a secure place and make sure you do not lose them Contents A 309 V Index
152. n can be used to move fluids through microfabricated channels Electrophoresis A standard technique of separating molecules on the basis of their mobility charge to mass ratios An electrical potential is applied across a capillary containing a sample in a fluid medium Positive molecules migrate towards the cathode and negative molecules migrate towards the anode at different speeds depending on their electrophoretic mobility Electrophoretic flow A macroscopic phenomenon that results from an electrical double layer formed by ions in the fluid and surface electrical charges immobilized on the capillary walls When an electric field is applied the bulk solution moves towards one of the electrodes cathode Electrodes sit in the reservoirs that connect to the ends of the various channels Electrode potentials are applied to the various reservoirs in a time dependent fashion to move the fluid in the required direction The gel filled channels of the LabChip devices do not exhibit a measurable flow because of dynamic channel coating and viscosity of the polymer matrix Contents A 346 V Index End Point The peak find algorithm looks for a leveling off when the value of the slope is less than the value set for the slope threshold This is considered to be the end point of the peak With RNA assays individual peak end times can be moved manually by dragging the diamond shaped end points shown in the single well display End Time This
153. n is complete NOTE The Auto Print settings are independent from those made via the Print command of the File menu 4 In the Analysis section you can activate the Pause Analysis on Setpoint Change function If this function is not active the measurement results are recalculated every time after you change a Setpoint If you need to change several setpoints at once activating this function saves you time because the results are only recalculated when leaving the setpoint explorer or when starting the analysis manually with the start button 5 Inthe Graph Signal Color section click the colored rectangles to the right of the signals You can now choose a new color for the selected signal in the Color dialog box Contents A304 V Index How to Set Auto Export Options To define auto export options 1 Switch to the System context and select the System Wide Settings tab 2 Select Auto Export in the tree navigation The Auto Export screen becomes visible J Auto Export Electrophoresis Export Result Tables Create a csv file containing result table values I Exclude Markers Include Ladders Strip off Excluded Peaks IV Sample Data Export textual list files one file per sample IV Gel Image Export the gel image IV As one image fa Formats J Each lane separately Formats IV Electropherogram Images an Formats Export the electropherogram of each sample as a separate file Flow Cytometry
154. n peaks Detailed descriptions of the available functions as well as the required syntax and examples are shown in the Help field at the bottom of the screen NOTE If the entered syntax is not correct the invalid part is displayed in red color 5 Click the Edit button next to the Rule Condition field and enter the logic expression for this rule As an example for a logic expression for the rule condition enter NumberOfPeaks gt 9 AND PeakFoundAuto 150 With this rule all samples can be found that have more than nine peaks while one of them Is located at 150 bp 10 6 Click the Edit button next to the Rule Comment field and enter a comment for this rule 7 Click the Edit button next to the Rule Color field and select a color for the rule If you check the Gradient check box you can assign a color gradient to the rule This allows to imply further information in the displayed results Contents A 162 V Index For example the rule condition NumberOfPeaks gt 0 marks all samples with peaks If you want to indicate the actual number of peaks with the color code you need to enter NumberOf Peaks in the Rule Color field Then you define light green for the Minimum Value 1 and dark green for 10 peaks as the Maximum Value As a result a darker green will be displayed for samples with more peaks 8 If necessary generate additional rules Rearrange the order of the rules with the Move Selected Rule Up and Move Selected Rule Down
155. nalyzer has been detected Check the COM Port setting see figure under step 3 the RS 232 connection cable the power cable and the power switch For details on how to set up the bioanalyzer and connect it to a PC see Agilent 2100 Bioanalyzer Installation and Safety Guide 2 Make sure that a bioanalyzer has been detected before continuing Contents A18V Index 3 Select an assay for the chip run On the nstrument tab click the Assays button 0R Click the Assays menu Both will open a menu allowing you to select an assay for the measurement Note that you can also select File gt Open File to Run This opens a dialog box allowing you to load either an assay xsy or a chip data file xad 4 Prepare the samples and the chip For detailed information on sample and chip preparation refer to Reagent Kit Guides that are available for each reagent kit Application Notes that are available for specific kits and applications NOTE When preparing chip and samples pay attention to the essential measurement practices described in Essential Measurement Practices Electrophoretic Assays on page 55 and Essential Measurement Practices Flow Cytometric Assays on page 187 or as described in the respective Reagent Kit Guide Contents A19V Index 5 Insert the chip in the Agilent 2100 bioanalyzer a Open the lid The status of the bioanalyzer is updated on the nstrument tab b Check that the cartridge
156. necessary markers and regions for evaluation The gating direction for histograms is given for details on the gating direction refer to Gating on page 214 The markers in all samples are connected changing a marker changes the corresponding markers in all samples Contents A 177 V Index The regions of the dot plots are related to the markers of the histograms Thus the results of the dot plots are identical to the results of the histograms Generic assay This assay has no specific settings and can be used to define individual assays You can freely add markers or regions and define the gating direction The generic assay is recommended for chips with different samples and stainings where regions would need to be defined individually Dot plot and histogram regions are not linked making it possible to evaluate an individual sample with different settings Flow cytometry assay icons On the Assay Properties tab the following icons are used to visualize the assay type Antibody Staining Apoptosis GFP Generic Blue to Red Red to Blue Checkout Beads siRNA Transfection Viability Contents A 178 V Index Preparing and Running a Flow Cytometric Assay A flow cytometric chip run requires the following steps 1 Switch on the Agilent 2100 bioanalyzer and start the 2100 expert software Details are given in Starting 2100 Expert on page 31 Select a flow cytometric assay See Selecting a Flow Cytometric Ass
157. need to know the following e Principles of Nucleic Acid and Protein Analysis on a Chip on page 46 e Preparing and Running an Electrophoretic Assay on page 50 e Analyzing and Evaluating the Results of an Electrophoretic Assay on page 81 e Result Flagging on page 152 Contents A4V Index Principles of Nucleic Acid and Protein Analysis on a Chip The electrophoretic assays are based on traditional gel electrophoresis principles that have been transferred to a chip format The chip format dramatically reduces separation time as well as sample and reagent consumption The system provides automated sizing and quantitation information in a digital format On chip gel electrophoresis is performed for the analysis of DNA RNA and proteins The chip accommodates sample wells gel wells and a well for an external standard ladder Micro channels are fabricated in glass to create interconnected networks among these wells During chip preparation the micro channels are filled with a sieving polymer and fluorescence dye Once the wells and channels are filled the chip becomes an integrated electrical circuit The 16 pin electrodes of the cartridge are arranged so that they fit into the wells of the chip Each electrode is connected to an independent power supply that provides maximum control and flexibility Charged biomolecules like DNA Contents A46V Index RNA or protein LDS micells are electrophoretically driven by a volta
158. ng lever in the open position as shown in the following figure CAUTION Do not touch the electrodes while the cartridge is in the Agilent 2100 bioanalyzer The electrodes and the high voltage power supplies can be damaged Metal lever in open position co 7 S The cartridge is pushed out Contents A 190 V Index 2 Gently pull the cartridge out of the lid CAUTION Improper handling of the electrode cartridge will damage it Always store the electrode cartridge in the provided box If the pins of the electrode cartridge are bent or misaligned poor quality results or pre terminated chip runs will result 3 Slide the pressure cartridge in the lid as shown in the following figure Push here to ensure tight connection Metal lever IN A A 4 Push the metal front of the cartridge until it is securely in place 5 Push the metal locking lever in the flat closed position Contents A 191V Index To load the prepared chip into the Agilent 2100 bioanalyzer 1 Open the lid and remove any chip 2 Adjust the chip selector to position 2 as shown in the following figure To avoid using incompatible chips and cartridges a chip selector is installed in the bioanalyzer This ensures that the chip matches to the installed cartridge This will allow you to insert cell chips in the bioanalyzer
159. nserted marker If you use a predefined assay the entry can be Annexin V on subset for example Minimum fluorescence value of the corresponding marker Maximum fluorescence value of the corresponding marker Number of events covered by the marker For the histogram you use for gating the number of all detected events is displayed in the row All events of selected events in relation to the total number of events The marker used for gating is 100 the table of the gated histogram shows the value of the subset of the gated events in relation to the total number of events These are the events that have passed the gate and are covered by the marker of the histogram for example by annexin V Mean fluorescence value of the events inside the marker Standard deviation to the mean value Coefficient of variation Geometric mean Contents A232 V Index Using Dot Plots for Evaluation On the Dot Plot tab cells are displayed as dots where their red fluorescence intensity is mapped on the Y axis and their blue fluorescence intensity is mapped on the X axis NOTE The lower left region of the dot plot area may show no events because of the threshold for event detection Dots are only displayed if their fluorescence intensity exceeds a minimum limit The limits are specified in the assay separately for red and blue fluorescence To evaluate the dot plots you can add regions Regions are rectangles that can be
160. nt 2 Instrument 3 Name COM Port Joemo 4 Serial Cartridge Method Selection be Methods Start Stop Run 5 Start Product ID Method C Demo DNA 7500 Custom xsy Firmware Simulation Mode Data File lt e S a 3 Assay Comments omparison T paanan Copyright 2003 Agilent Technologies sS R Default C sDNA DNA 7500 Demo DNA 7500 Custom Method Custom C sDNA DNA 7500 Demo DNA 7500 Custom File Prefix 100 expert max 16 characters System A Data Acquisition Parameters Start Run Checklist Is the instrument ready Run sample 1 f2 v Ei a of Isa chip detected of Is selected instrument valid for this assay of Does the cartridge and the selected assay match of Are all required licenses applied of Is current instrument valid for the selected method of Does the user have rights to start a run Chip Setup I offline Demo Mode Mr Advanced as Advanced Operator Auto Export Auto Print NOTE If you started 2100 expert for the first time after installation you first need to activate the different software modules with your license keys See Figure How to Activate Software Licenses for details Contents A1l1V Index The nstrument tab shows you the status of the bioanalyzer Icons Meaning Bioanalyzer detected lid is open Bioanalyzer detected Lid is closed but no chip Is inserted No bioa
161. nt along the line of the fragment until it is positioned as desired Sample 7 Move any other start or end points as desired Contents A123 V Index TIP The fragment table can be directly edited in the setpoint explorer Name From s 2 To s Color Pies 38 5 39 72 2 235 43 56 419E NOTE Changing the start or end points of the fragment will change the calculated rRNA ratio It might be convenient to pause the automatic analysis E ectropherogram gt Pause Automatic Analysis until all changes are done Setting the Baseline for Calculation of RNA Concentration At low signal to noise ratios the baseline that defines the area used for calculating the concentration of RNA assays is highly dependent on the settings for the Start and End Time You can adjust the Start and End Times manually thereby adjusting the baseline Contents A124 V Index to ensure a good result even at very low signal to noise ratios Choose a single sample Two vertical green long dashed lines indicating the setpoints for the Start and End Times with the baseline drawn between them are displayed in the window Sample 7 I Move the cursor over the long dashed line on the left Start Time setting and drag the line to the desired position Do the same with the long dashed line on the right End Time setting until you have a flat baseline NOTE Changing the start and end times will change the
162. o Configure Markers on page 222 Contents A 224 V Index How to Copy Markers to All Histograms Once a marker is defined you can copy it in the histograms of all samples generic assays only 1 Select the marker in the histogram or in the result table The nsert the selected marker into all histograms button Sis now enabled 2 Click this button The Copy Marker dialog box appears This dialog box asks you whether or not you want to use the marker as reference 3 Click Yes to use this marker as reference The marker will be inserted in all other histograms of the blue or red channel When the properties of this marker are changed the changes will be applied to all samples 0R Click No The marker will be inserted in all other histograms of the blue or red channel When the properties of this marker are changed the changes are only applied to the current sample How to Set the Gating Direction Generic assay only You can use one marker to define the gating direction In other words you define whether red or blue fluorescence is used as a gate to define a subset in the other histogram This also depends on the dyes that you have used for staining You can set both gating directions either from the blue histogram to the red histogram or from red to blue Contents A 225 V Index To set the gating direction 1 Select the marker in the red or blue histogram you want to use as a gate for the other histogram
163. of popular bitmap graphics formats Tiffs are commonly used in DTP work because of their support for color specification Contents A 357 V Index U Upper Marker An internal standard that is added to a DNA or Protein sample in a well to assist in determining size and concentration of the sample The upper marker is the same as the last peak found in the sizing ladder Ww WAV file A type of computer file used to store a sound digitally Workflow The workflow defines the order of steps that need to be taken for a measurement to ensure data validity and data reliability This includes steps such as the execution of methods result reviews and the final approval The workflow definition is part of the methods and is available in the 2100 expert software only with the security pack installed WMF file Windows Metafile Windows metafile documents can contain any mix of vector and raster or bitmapped information to describe the contents of an image WMF graphics are generally used on the Windows platform as a standard format for clip art and other graphically rich information such as charts Contents A 358 V Index X XAD file 2100 expert chip data file The files contain raw data assay information data analysis setpoints information on chip samples and study and the run log information XAC file 2100 expert comparison file XLS file Microsoft Excel spreadsheet file XML file Extensible Markup Language files
164. oint to point fit For each sample peak the center time is interpolated from the Standard Curve to determine the peak size in base pairs Start Point The peak find algorithm walks the data from time zero looking for a slope greater than the Slope Threshold This is considered to be the start point of a peak With RNA assays individual peak start times can be moved manually by dragging the diamond shaped start points shown in the single view Contents A 356 V Index Start Time This setting determines the time after which the first peak or fragment will be located any peaks appearing before this time are ignored In RNA and Protein assays the start time is shown on the single view display as a long dashed vertical green line note that this is true for protein assays when analysis is on the start time is shown as a solid green line when analysis Is off for protein assays With RNA assays another start time setting is available that determines the start time for an individual peak With RNA assays individual peak start times can be moved manually by dragging the diamond shaped start points shown in the single view T Tool Tip A small box containing text that describes the item indicated by the mouse pointer To view a Tool Tip position the mouse pointer over an object on the screen Leave the mouse stationary for a moment and a Tool Tip if one exists for that item will appear TIF file A file extension indicating one of a set
165. on of the current sample will be aborted The following message appears 2100 expert x x re you sure you want to stop the run 2 Click Yes to stop the chip run When the chip run is aborted you can e Switch to the Data context where you can view analyze and evaluate the results if any of your chip run see Displaying the Measurement Results Flow Cytometry on page 205 and Analyzing and Evaluating the Results of a Flow Cytometric Assay on page 211 e Stay in the nstrument context where you can start the next chip run for example Contents A201 V7 Index Entering Chip Sample and Study Information During or after a chip run you can document the run by entering information on chip samples and study 1 In the Data context select the Chip Summary tab 2 On the Sample Information sub tab you can enter additional information for samples such as names for blue and red stain On the Study Information sub tab you can enter information such as the name of the current study the laboratory location and the experimenter Contents A202 V Index gt File Name 2100 expert_APOPTOSIS_00000_2003 08 01_18 27 23 xad Location C Program Files Agilent 2100 Bioanalyzer 2100Xpert data 2003 08 01 Created Friday August 01 2003 6 27 27 PM Modified Wednesday August 06 2003 10 44 05 4M Software Created by version B 01 00 51052 modified by 4 00 00 51000 TY Agilent Technologies C
166. oolbar The Configure Region dialog box appears Configure Region 3 x Name Region 1 Top 3438 99 Left 6 88 Bottom 21 4 Right 300 58 Region Color Ea a Cancel Help 2 Enter a Name for the region It is advisable to use an easy to understand name Contents A 236 V Index 3 Enter fluorescence values for the left right bottom and top side of the rectangle to define position and size of the region These values correspond to the upper and lower marker limits of the blue and red histograms 4 Click the button next to the color square to open the Color dialog box and select a color for the region border 5 Click OK To color dots inside regions 1 Click the Color Dots button in the toolbar All dots inside the regions now have the same color as the region border In case of overlapping regions dots are colored with the color of the last added re positioned or resized region Contents A 237 V7 Index How to Change Position and Size of a Region You can change the size and position of regions to restrict the number of included events You can work graphically with the mouse or enter the values in the Configure Region dialog box To move a region 1 While pressing the Shift key click the region border and drag the region to the new position 2 Release the mouse button To change size and position with the mouse 1 Click the region border to select the region 2 Position
167. or one sample depends on the number of samples that are measured per chip If only one sample is measured you can set the run time up to 1440 s Contents A 184V Index Preparing Samples and Chips for Flow Cytometric Assays WARNING Several substances such as dyes can have toxic carcinogenic or mutagenic potential Therefore carefully follow the safety instructions from the dye safety data sheet and the Reagent Kit Guides Also read the Essential Measurement Practices Flow Cytometric Assays on page 187 Before you can fill a chip you have to prepare the samples To find out which protocols you should use to prepare the samples refer to the various Application Notes available for each assay Sample and chip preparation is described in detail in the Reagent Kit Guide available for each LabChip kit Contents A 185 V Index Chip Reagents Several reagents have to be added to the chip to prepare it for measurement The following image shows which reagents have to be filled in which wells Priming solution Cell buffer Sample 1 6 Cell buffer gt Agilent Teclmolog ies Focusing dye solution Make sure you follow these directions when preparing the sample e The priming solution has to be added first It fills all channels removes all air from the micro channels vertically before each chip is measured The focusing dye is used to adjust the optic The optics are focused horizontally and e If you
168. orescence to obtain percentual values on a defined population Contents All4V Index In predefined assays the borders of the rectangular region represent the markers defined in the corresponding blue and red histograms Gate Region 1 10 Red Fluorescence Torrrent rere rT Q 1 TH 1 LLY 1 LLL 1 LHL sl ee 1 boai SS TARR O EA 107 10 10 10 108 Blue Fluorescence b The lower left region of a dot plot usually shows no events due to the defined peak detection threshold that the fluorescence values must exceed For detailed information see Using Dot Plots for Evaluation on page 233 Contents A 175 V Index Overview of Flow Cytometric Assays The cell characteristic to be measured requires not only specific dyes Several measurement parameters to control the measurement and the data acquisition parameters also have to be specified These so called setpoints are stored in assay files xsy and are read by the 2100 expert software before it starts the measurement 2100 expert supports the following assays based on flow cytometry Predefined assays e Apoptosis e Apoptosis fast protocol For reduced background this assay has an increased threshold and uses blue events only for peak detection Antibody Staining On chip Antibody Staining For reduced background this assay has an increased threshold in the blue signal e GFP On chip GFP This assay all
169. ot Reviewed iles General Properties Assay Properties Chip Summary Gel Electropherogram Result Flagging Log Book E E 2100 expert_DNA 7500_001 E All Samples FU PCR Mix 3a C PCR Mix 1 O Sample 3 C Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 Sample 12 Ladder Verification 1e 5 Oooo Comparison O SS Method System 50 300 500 700 1000 2000 10380 bp a Size bp Conc ng l Molarity nmolfl Observations F 1 50 8 30 251 5 Lower Marker 2 74 8 80 179 7 3 113 1 92 25 7 ies es a a a a ec 4 165 0 95 8 7 j 215 2 56 18 0 2 2 2 21 221 ji 261 1 55 9 0 T i 310 1 98 9 7 E 8 417 2 94 10 7 sas ese u 514 7 66 22 6 hal t illa 456 7 8 9 101112 Besults Peak Table Legend Errors mr Advanced as Advanced Operator auto Export Auto Print The measurement data is stored in xad files Contents A3 V Index Verification Context The Verification context is used to run and document qualification tests 322100 expert C ilent 2100 bioanalyzer 2100 expert SecuredArea Validation erification_19 05 2005_14 32 09 xvd Eile Context View Windows Help Active Verification ification_19 05 2005_14 32 09 xvi
170. ould start with the most specific rule If that one does not apply a less specific one may apply If none of the defined rules apply the final default rule defines the color code In Jarget mode all rules are applied subsequently If the next rule applies the color code changes to the color code defined by the rule otherwise the previous color code is kept Therefore the last matching rule defines the color code of the sample This mode is called target mode because later rules refine the result color code The first default color code is the most general and the last one the most specific You can define the flagging rules already in the assay or after the chip run is finished modify these rules or define new rules in the chip data file and apply the rules to the measurement results Defined rules can also be saved loaded and applied to other data files Contents A 153 V Index TIP The examples shown in this chapter are taken from the demo assay Demo Protein 200 Plus xsy that comes with the 2700 expert software You can find this demo assay in the assays demo electrophoresis subdirectory of the 2700 expert installation folder In the data samples resultflagging subdirectory of the 2700 expert installation folder you can find further examples for result flagging rules xml which you can import in the Protein 200 Plus demo assay Contents A154 V7 Index Defining Result Flagging Rules The rules
171. ove them from your hard disk Depending on the amount of hard disk space available to the 2100 expert software you may need to clear space on your hard drive to ensure that you will have enough room to save upcoming chip run data Contents A 270V Index Importing Data When working with assay xsy or chip data xad files you enter specific information that you may want to reuse To support the reuse of data 2100 expert has the following import capabilities e Importing Bioanalyzer Files on page 2 2 e Importing Data Analysis Setpoints on page 273 e Importing Chip Sample and Study Information on page 275 You can import result flagging rules definitions for result flagging into electrophoretic assay or chip data files e Importing Result Flagging Rules on page 276 Contents A 271V Index Importing Bioanalyzer Files You can import data assay and method files that were generated with other Agilent 2100 bioanalyzer systems You can even import data and assay files from the older Bio Sizing and Cell Fluorescence software applications To import assay files 1 Switch to the Assay context 2 From the File menu select mport to display the Open dialog box 3 Select a file of any of the following types xsy 2100 expert assay file asy Bio Sizing assay file csy Cell Fluorescence assay file 4 Click Open The imported file appears in the Tree View Panel and the Assay Properties tab sh
172. ows information about the assay Upon importing the file gets converted to a new 2100 expert assay file xsy To import chip data files 1 Switch to the Data context 2 From the File menu select mport to display the Open dialog box 3 Select a file of any of the following types xad 2100 expert assay file Contents A212 V Index cld Bio Sizing assay file cad Cell Fluorescence assay file 4 Click Open The imported file appears in the Tree View Panel and the electropherogram grid view shows an overview of all samples Upon importing the file gets converted to a new 2100 expert chip data file xad Importing Data Analysis Setpoints You can import data analysis setpoints from other assay xsy or chip data xad files of the same type Note the following when importing e Electrophoresis files to be imported must be of the same assay type This means that you Cannot import setpoints from a DNA 1000 assay into a DNA 500 assay for example e Flow cytometry files to be imported can be of any flow cytometric assay type but the import will change the type of the current file to Generic To import data analysis setpoints 1 On the Assay Properties tab click Import Setpoints 2 The Open dialog box appears 3 Select the file from which you want to import and click Open Contents A213 V Index NOTE For flow cytometry files the import will de ete all existing markers and regions in the cur
173. ows a rapid and accurate detection of green fluorescent protein expression Blue to red This assay is for applications that apply a blue reference dye and analyze red fluorescent cells within a blue population Contents A 176 V Index e Red to blue This assay is for applications that apply a red reference dye and analyze blue fluorescent cells within a red population Checkout Beads Red checkout beads are loaded into the wells 1 3 and 5 and blue checkout beads into the wells 2 4 and 6 Markers are set according to expected fluorescence levels of the red and blue beads The Checkout Beads assay has the properties of a generic assay see below siRNA Transfection Viability Transfection Viability analysis as described in the Application Note s RNA transfection optimization with the Agilent 2100 bioanalyzer Agilent publication number 5988 9782EN This assay enables the automatic calculation of transfection efficiency TE in histogram view and viability in transfected cells ViT in dot plot view Required gating directions and regions are provided as example but can be adjusted Final Transfection Viability TV can be calculated by multiplication TE and ViT values derived from histograms and dot plots The settings of predefined assays are optimized to measure the appropriate cell characteristics For evaluation it is only necessary to adjust the markers in histograms or regions in dot plots Predefined assays contain all
174. r making changes it will keep a record of the changes such as gel color sample names and peak find settings that were in effect at the time the file is resaved If you do not want to change the original file choose File gt Save As and give the file a new name or save it to a different location Contents A142 V Index Comparing Samples from Different Electrophoretic Chip Runs The 2700 expert software allows you to compare the measurement results of samples from different electrophoretic chip runs Samples to be compared must be from chip runs of the same assay type In the Comparison context you can create comparison files include samples from different chip runs and compare the samples by overlaying electropherograms for example To compare samples from different electrophoretic chip runs 1 Switch to the Comparison context 2 From the File menu select Open and open all chip data files xad that contain the samples you want to compare The xad files appear in the Select Data Files list of the Tree View Panel NOTE The Select Data Files list also contains all electrophoretic xad files that are currently open in the Data context Contents A143 V7 Index 3 Select a xad file from the Select Data Files list to display a list of its samples Select Data Files 2100 expert_Protein 200000 M 2100 expert_DNA 1000_00000 Protein Ladder 3 Protein Ladder 2 IgG nonreduced Protein Ladder 3 4 Right click a sample name
175. red histogram the blue fluorescence values to the marker in the blue histogram As in the histogram evaluation high blue fluorescence and high red fluorescence mean living cells with bound antibodies See the following example Cumulation of high blue and high red fluorescence indicates strong binding of labeled antibodies in living cells CD3 APC Red Fluorescence 3 LLL JL y L LL Loiri 10 10 10 10 10 10 Blue Fluorescence Contents A 246 V Index The results of the dot plot evaluation are numerically displayed in the result table Events covered by the region All measured events oem 8 Events Z total Z of gated Amount of living cells in relation to all measured cells Amount of living cells with high antibody binding in relation to all living cells Evaluating Apoptosis Assays The apoptosis assay can be used to examine how many apoptotic cells are within a living cell population Dead or necrotic cells can be excluded from the evaluation For a detailed description on how to evaluate the results using histograms and regions refer to Using Histograms for Evaluation on page 212 and Using Dot Plots for Evaluation on page 233 Living or dead cells In most cases you want to know whether cells are dead or alive at a specific time For this you can use calcein AM as living cell dye for example This dye accumulates in intact cells whereas it will leak out of damaged cel
176. rent file and change the current assay to a Generic assay A message box appears that prompts you to confirm this change Import Marker and Regions xi Assay will be changed to GENERIC Do you want to continue 4 Click Yes NOTE Importing data analysis setpoints overwrites all current setpoint values All files the setpoint values are updated in the setpoint explorer and immediately applied to the measurement results if any Flow cytometry files the new markers and regions are now available for evaluation and calculations based on the new markers and regions are immediately done 5 From the File menu select Save to make the changes permanent Contents A214 V Index Importing Chip Sample and Study Information On the Sample Information and Study Information sub tabs of the Chip Summary tab you can enter names and comments regarding chip samples and study The information you enter here may be very similar for further chip runs or other assays Once you have entered the information you can export it into a separate file see Exporting Chip Run Data on page 278 which you can then import into other chip data xad or assay xsy files instead of typing it anew The import export files can have the extension txt or csv and have a fixed form which differs for electrophoretic and flow cytometric assays To import chip sample and study information 1 On the Chip Summary tab click Import 2 The
177. retic Chip Runs on page 143 How to Use the Form Mode on page 159 Contents A81V Index Data Analysis DNA The data analysis process for DNA assays consists of the following steps 1 Raw data is read and stored by the system for all of the individual samples 2 The data is filtered and the resulting electropherograms of all samples are plotted You can change the settings of the data analysis after the run and reanalyze your data 3 Peaks are identified for all samples and are tabulated by peak ID You can change the settings of the peak find algorithm and reanalyze the data after the run has finished Note that peak find settings can be changed for all or only certain samples Contents A82V Index 4 A sizing ladder see the following example electropherogram which is a mixture of DNA fragments of known sizes is run first from the ladder well The concentrations and sizes of the individual base pairs are preset in the assay and cannot be changed FU Ladder 50 WTI Size bp Conc na ul Molarity nmol l Observations gt 15 4 20 424 2 Lower Marker 50 2 00 60 6 Ladder Peak 25 2 00 121 2 Ladder Peak Contents A83V Index 5 A standard curve of migration time versus DNA size is plotted from the DNA sizing ladder by interpolation between the individual DNA fragment size migration points The standard curve derived from the data of the ladder we
178. rocess of finding a particular file when someone wishes to examine the data again Even if only one person uses the 2100 expert software it is still wise to review your files periodically archive files you are no longer using but wish to save and discard unneeded files Organizing 2100 Expert Data Each user in your laboratory may want to specify a particular prefix that will easily differentiate their data files from any others To do this switch to the System context go to the System Wide Settings tab and select Data Files in the tree navigation Then activate the Prefix check box and edit the prefix string as you require Note that you can also modify the file prefix before you start a chip run Additionally you may specify that a new directory is created each day for storage of that day s runs To do this activate the Create Daily Subdirectories check box on the same screen Backing up 2100 Expert Data It is strongly recommended to save your files to a backup disk or on CD DVD on a regular basis This allows to retrieve the data in case of a system crash or other cases of data loss Contents A 269 V Index Archiving 2100 Expert Data The difference between archiving and backing up Is that in the archiving process the data will be removed from its original place and moved while during the backup process only a copy is taken depending on the tools you use It is a good idea to periodically archive your files to a CD DVD to rem
179. rors Index How to Navigate Through the Samples At any time even during a chip run you can scroll through all samples either in electropherogram or gel view To navigate through samples using the Tree View Panel 1 If the tree view is not visible select View gt Tree View The tree view panel appears to the left of the tabs and shows all chip data and assay files as nodes 2 Click any sample name Electropherogram view the electropherogram of the selected sample is shown in single view Gel view the lane of the gel like image corresponding to the selected sample is highlighted To navigate through samples using the Lower Panel 1 If the lower panel is not visible select View gt Lower panel The lower panel appears in the lower left corner 2 Electropherogram view Click any lane of the small gel image Gel view Click any well on the chip icon Contents ATAV Index To browse through samples 1 From the Electropherogram or Gel menu select Next Sample or Previous Sample 0R Click the Next Sample or Previous Sample button in the toolbar To switch between electropherogram and gel view 1 Click the Electropherogram or Gel tab to display the results of the selected sample as an electropherogram or as a gel like image How to Change the Display of Electropherograms and Gel like Images It is possible to change the display of electropherograms and gel like images In electropherograms and gel like images you can
180. rt on page 300 for details e Log books are provided that record all important actions and messages in the 2100 expert software See Using Log Books on page 310 for details Contents A 299 V Index Configuring 2100 expert The available options for configuring the 2100 expert software can be found in the System context on the System Wide Settings tab How to Specify Data File Names and Directories The measurement results are stored automatically when the chip run is complete To make it easier for you to identify the chip data files you can configure an automatic naming scheme for the files Contents A 300 V Index To specify the names and destination for generated chip data files 1 Switch to the System context and select the System Wide Settings tab 2 Select Data Files in the tree navigation The Data Files screen becomes visible Data File Name Create Data file names by combining IV Prefix fat 00 expert IV Assay Class IV Serial Number V Date Time Example 2100 expert ASSAYCLASS_SRNUM_2005 04 29_ C Counter Data File Directory Default Directory E 4 42100 bioanalyzer 2100 expert Data Browse Reset IV Create Daily Subdirectories Data File Format Binary Format XML Format 3 Inthe Data File Name section select the check boxes of the strings you want to insert in the file names Option Meaning Prefix Inserts an arbitrary string to identify the data file This
181. s NOTE Raw data acquired from the bioanalyzer is not changed only evaluation and display of the results can be changed and saved If you alter the data shown in any way after it has been saved and try to exit the program or switch to a different context to acquire new data for example a dialog box will appear asking whether or not you wish to save the changes Opening chip data files as read only A chip data file can be opened as read only the Title Bar will show Read Only at the end of the filename The read only file can be edited but may not be saved under the same name If you attempt to save an edited read only file and error message will be displayed explaining that the file is a read only file Contents A 2607 V Index The benefit of opening chip data files as read only is to prohibit you or other users from making changes that would alter the file in any way Because the 2100 expert software allows you to open chip data files modify data and save them you may prefer to ensure that the original parameters that were used to create the file are not altered Contents A 268 V Index Organizing Backing up and Archiving 2100 Expert Data As you begin to work with the 2100 expert software it is good practice to organize your files If you are not the only user of the bioanalyzer creating a directory within which to save your files is recommended having each person save files to their own directory will speed the p
182. s you like The following values are displayed Region The default region Al Events is always displayed in the first row and shows the values for all detected events For each further region see How to Add Regions to Dot Plots Generic Assay only on page 234 a row is added to the table XMean Mean fluorescence values in x direction YMean Mean fluorescence values in y direction Events Number of events for each region added to the dot plot Total Percentage of events for each region added to the dot plot of gated Percentage of the gated events in the region StdDevX Standard deviation to the mean fluorescence value in x direction StdDevY Standard deviation to the mean fluorescence value in y direction CV X Coefficient of variation of the x values CV Y Coefficient of variation of the y values X GMean Geometric mean of the x values Y GMean Geometric mean of the y values Contents A2MV Index Evaluating Antibody Staining Apoptosis and GFP Assays With the 2100 expert software several predefined assays are supplied You should only use each assay for the specific experiment for which it was developed For example you have to use the read dye for detection of apoptosis calcein and Cyd for example e Evaluating Antibody Staining Assays on page 243 e Evaluating Apoptosis Assays on page 247 e Evaluating GFP Assays on page 253 Contents A242 V Index Evaluating Antibody Staining Ass
183. splay details for example e puta color gradient on the background of the graphs In histograms you can additionally e show data points To zoom into a histogram or dot plot 1 Position the mouse pointer in the histogram dot plot 2 Click and hold down the left mouse button The mouse pointer changes its shape to a magnifying glass Qo 3 Drag the mouse A rectangle shows the part of the histogram dot plot to be enlarged 4 Release the mouse button Contents A 208 V Index Annexin C 5 Annexin C 5 1 24h fix Calcein 1 24h fix Calcein on oa Annexin p O No of Events w ao 10 2 Fluorescence z Fluorescence al g g You can perform several zoom steps in a row When you have zoomed a histogram or dot plot the Undo Zoom and Undo All buttons are enabled To undo one zoom step 1 Click the Undo Zoom button or double click in the histogram or dot plot To undo all zoom steps 1 Click the Undo Zoom All button Contents A 209 V Index To display data points in histograms 1 From the Histogram menu select Show Data Points All events are shown as bullets To put a color gradient on the background of a histogram or dot plot 1 From the Histogram or Dot Plot menu select Gradient 0R Click the Gradient button the histogram or dot plot toolbar A color gradient gray to white appears on the background of the graph Annexin C 5 1 24h fix Calcein Annexin
184. ssessecseeeeceeseseecaeseesecaeensnesseeaseansneaess 316 How to Run Instrument Diagnostics Te Sts cccccsescscescsesssscscsescsessesssecsescssseseseeesaes 321 Performing Verifications ros ois conceiienienctvorterntuperstmnanieticwstlavdede avatar eaedt 325 Products Spare Parts and ACC SSOFICS c ccscsccsesesecseseesessseeeeseeseeseesessaesaesaeeaes 334 NSS ANY cess scoceek oie sce eannan ensedar draa secs czciesssccsecxcescesceccasbesecssecececcnsecccet cosseetesscrcceeseceeaderases 338 Contents A4YV Index About this Manual Welcome to the User s Guide for the Agilent 2100 expert software This manual provides beginners and advanced users with information needed to successfully run electrophoretic and flow cytometric assays with the bioanalyzer The 2100 expert software allows the control of the bioanalyzer including diagnostic functions and in combination with a LabChip kit the acquisition interpretation and result presentation of data generated during the analysis of DNA RNA proteins and cells Contents A5V Index In this Manual This manual provides bioanalyzer users with the following information e About this Manual on page 5 gives an overview of the subjects in this manual and lists major innovations and improvements of the 2100 expert software It also lists supplemental literature and shows you how to make efficient use of this manual e Quick Start on page 14 is meant for experienced us
185. ssories Cartridge and license must be purchased separately G2943CA Agilent 2100 bioanalyzer laptop system Includes Agilent 2100 bioanalyzer HP Compag laptop PC color printer system software vortexer and accessories Cartridge and license must be purchased separately Contents A334V Index Hardware e G2938C Agilent 2100 bioanalyzer Includes 1 chip priming station 1 test chip kit serial cable nstallation and Safety Manual Cartridge and license must be purchased separately G2947CA Agilent 2100 bioanalyzer electrophoresis set Includes test chip kit electrode cartridge license key for electrophoresis assays and start up service G2948CA Agilent 2100 bioanalyzer flow cytometry set Includes checkout kit test chip kit pressure cartridge license key for flow cytometry assays and start up service Software and Services e G2946CA Agilent 2100 expert software upgrade Software package for upgrade to the latest 2100 bioanalyzer system software revision e G2949CA Agilent 2100 expert security pack Additional services for Installation Qualification IQ and Operation Qualification Performance Verification 0Q PV as well as assay consulting are available and can be ordered separately Contents A 335 V Index Spare Parts and Accessories 5065 4413 Electrode cartridge 5065 4492 Pressure cartridge 5065 4478 Pressure Adapter Kit Contains 5 plastic adapters and 1 mounting ring for use w
186. step that occurs after samples are loaded into the chip is designed to rid the wells of bubbles and is usually very effective If a large bubble is seen at the bottom of a well remove the sample from the well pipette it back in and continue with the loading procedure Contents A343 V Index C CAD file In Cell Fluorescence flow cytometric chip runs were stored as cad files 2100 expert can import cad files See also XAD file Center Point After locating a start point the peak find algorithm looks for the first negative slope value and saves the previous point as the center If the value of the center point is less than the Minimum Peak Height the algorithm starts looking for a new peak CLD file In Bio Sizing electrophoretic chip runs were stored as cld files 2100 expert can import cld files See also XAD file COM Port See Serial port CSV file Comma separated variable file The simplest form of file for holding tabular data Data is listed in columns in a text file each value being separated by a comma Each new line represents a new set of data Import and export with Microsoft Excel is possible CSY file In Cell Fluorescence flow cytometric assays were stored as csy files 2100 expert can import csy files See also XSY file Contents A344 V Index D Data Filtering The first step 2700 expert takes in analyzing raw data is to apply data filtering Data filtering is done by means of a polynomial filt
187. string can be modified The default file prefix is 2100 expert Assay Class Inserts the assay class in the file name Examples DNA1000 GFP Apoptosis Contents A301 V Index Option Meaning SerialNumber Inserts the serial number of the Agilent 2100 bioanalyzer instrument used for the chip run Date Inserts the date of the chip run Time Counter Inserts the time of the chip run inserts an auto incremented 3 digit number In the Data File Directory section specify the Default Directory where the chip data files are to be stored Use the Browse button to select a directory or click Reset if you want to use the Data directory under the 2100 expert installation directory Optionally you can select the check box Create Daily Subdirectories if you want daily subdirectories to be created This option helps you to better organize your chip data files If selected a subdirectory is created for every day in which a chip run was started The name of the subdirectory has the format YYYY MM DD for example 2005 01 22 All chip data files generated on this day will be stored in this subdirectory In the Data File Format section select whether you want to save the data files in Binary Format or in XML Format Use the Prefix field to specify a default prefix for the created files This default prefix can be changed by every analyst in the nstrument context for each chip run Contents A 302
188. the Verification context 2 From the File menu select New 3 A New Verification item appears in the Tree View Panel 4 Under Cartridge Details click Select and specify details on the cartridge that is currently installed in the bioanalyzer Contents A 326 V Index 5 Under Configure 2100 Bioanalyzer HW Test Chips enter the test chips you will use for this verification New Verification All Verifications Configuration New Verification Inst Bi I Set _ Installation Verification COSHH System Verification Electrophoresis Set License 264958350 16147633 1378230307 Flow Cytometry Set License 315289990 16152224 1379278883 m Select 2100 Bioanalyzer Instruments and Cartridges Instrument Details Cartridge Details Serial Number DE34903221 Identifier 1 Friendly Name DE34903221 Product ID G2938C FW Revision C 01 048 Select Configure 2100 Bioanalyzer HW Test Chips Test Chip Type Lot Number Offset Intensity Autofocus Test Chip FO7CTF 270 410 Electrode Diode Test Chip F23CTE Cell Autofocus Test Chip EAQSE Contents A 327 V Index 6 Inthe Tree View Panel navigate to the test category you want to execute Select the category via nstallation System Verification Software Hardware PC name Bioanalyzer name Test Category NOTE To execute hardware tests HW branch the bioanalyzer must be properly connecte
189. ther context For example you can evaluate any chip data file in the Data context or compare samples in the Comparison context e f necessary abort the chip run by clicking on the Stop button All data that was collected up to the stop point will be saved Contents A2V Index Viewing the Measurement Results To view the results switch to the Data context The data file that has just been generated by your chip run is displayed The Chip Summary tab shows information on your chip data file and lets you enter comments regarding chip samples and study 2100 expert C ea Data dsDNA DNA 7500 Demo DNA 7500 Custom 2100 expert_DNA 7500_00000_ 2005 05 19 _14 01 59 xad File Context View Workflows Windows Help e b e Eis a All Samples Data File 2100 expert_DNA 7500_00000_2005 05 19_14 01 59 xad Sample 1 t Location C 2100 expert Secured4rea Data dsDNA DNA 7500 Demo DNA 7500 Custom Sample 2 i Sample 3 79 Created Mai 19 2005 14 02 05 Sample 4 3 amp Modified Mai 19 2005 14 04 07 Sample 5 Sample 6 Software Created by version B 02 01 51211 modified by B 02 01 51211 Sample 7 Sample 8 Sample 9 Sample 10 ODDOROOE File Version 1 Latest version Comparison Sample 11 Sample Comment Result Label
190. tic measurements the electrode cartridge is required y UV WY The electrode cartridge contains 16 electrodes that fit into the wells of DNA RNA and Protein chips Each electrode in the cartridge has an individual power supply All electrophoretic assays DNA RNA and Protein require an electrode cartridge The electrode cartridges will either have an engraved 1 at the front or will have no engraving at all Cartridges with a different number are not electrode cartridges Contents A 58 V Index If the bioanalyzer is set up for flow cytometric assays but you want to run electrophoretic assays proceed as follows 1 Open the lid and pull down the metal locking lever into the open position as shown in the figure below Metal lever in open position The cartridge is pushed out 2 Gently pull the cartridge out of the lid NOTE Store the pressure cartridge in the provided box Contents A59I V Index 3 Slide the electrode cartridge into the lid as shown below Push here to ensure tight connection Metal lever CAUTION Do not touch the electrodes while the cartridge is in the Agilent 2100 bioanalyzer The electrodes and the high voltage power supplies can be damaged 4 Push the metal front of the cartridge to ensure a tight connection 5 Push the metal locking lever into the fl
191. tion for protein and DNA assays only e Absolute quantitation for protein assays only These settings can be made before a new run is started or when reanalyzing a previously saved data file Contents A116V Index About the Setpoint Explorer The tool allowing you to modify the data analysis setpoints is the Setpoint Explorer The setpoint explorer is accessible from e Assay Properties Tab e Electropherogram Tab Single Grid View e Gel Tab Contents A 117 V Index On the Assay Properties tab the setpoint explorer is always visible and lets you modify setpoints globally for all samples Local Global advanced v Collapse li l Quantitation Concentration of upper 4 2 Concentration of lower 8 3 Sizing Smear Analysis Perform Smear Analysis C Baseline calculation Baseline start time s 26 Baseline end time s 94 Zero Baseline x Filter Settings Filter width s 0 5 Polynomial order 4 Baseline correction Perform Baseline Corre C Integrator Integration start time s 26 Integration end time s 94 Slope Threshold 0 5 Area threshold 0 5 Height threshold FU 8 Peak filter width s 0 5 Baseline plateau s 5 Width threshold s 1 Peak Filter polynom 6 Ladder Setpoints Baseline calculation Baseline start time s 26 Baseline end time s 94 Zero Baseline x Standard Curve Point to Point al l Defauts Help Contents A11
192. to have measured The total number of samples that can be measured varies with the type of assay selected With DNA and RNA Nano assays 12 samples may be run with RNA Pico assays 11 samples may be run and with Protein assays the maximum number of samples is 10 When preparing the chip see Preparing Samples Reagents and Chips for Electrophoretic Assays on page 54 keep in mind that you have to follow the sequence of the sample wells For example if you want to measure only 3 samples you have to fill the wells 1 2 and 3 of your chip Contents A53V Index Preparing Samples Reagents and Chips for Electrophoretic Assays Before you can load a chip you have to prepare the samples and reagents To find out how to prepare the samples and reagents refer to the various Reagent Kit Guides available for each LabChip kit Please refer to these documents for further information and analytical specifications In general preparing an electrophoretic assay involves the following steps Check that you have everything that Is listed in the appropriate Reagent Kit Guide Be aware that there can be small but important differences between the different assays even for the same type of molecules for example between DNA 1000 and DNA 7500 assays Make sure you are familiar with the essential measurement practices see next page Before running the first RNA assay decontaminate the electrodes Prepare all the reagent mixtures for examp
193. to the following results Sample Name Sample Comment Use For Calibration Status Observation Result Label Result Color beta LG r Conc pg ml 0 Any peak at 30 kDa 7 0 1 2 betaLG 3 betaLG r 0 y y y Number of peaks between 5 and 10 Contents A 164V Index For sample 1 rule 1 matches and defines the color Rule 2 would also match but is not checked because the procedure stops at the first match For sample 2 none of the rules match if there is no peak at 30 kDa 7 Therefore this sample will get the default color For sample 3 only rule 3 matches and defines the color Contents A 165 V Index Running and Evaluating Flow Cytometric Assays For running and evaluating flow cytometric assays you need to know the following e Principles of Flow Cytometric Measurements on page 167 e Overview of Flow Cytometric Assays on page 176 e Preparing and Running a Flow Cytometric Assay on page 179 e Analyzing and Evaluating the Results of a Flow Cytometric Assay on page 211 Contents A 166 V Index Principles of Flow Cytometric Measurements Besides electrophoretic assays DNA RNA and proteins the Agilent 2100 bioanalyzer supports flow cytometric assays e First cells are stained with two fluorescent dyes that correspond to biologically relevant parameters as described in the application notes available for eac
194. to the samples by clicking the Apply Result Flagging icon A All samples are re evaluated according to the result flagging rule and displayed with the respective colors See Color Indication on page 157 for more information on the color codes Additional information is available in the Help panel at the bottom of the screen This panel provides context specific help including examples How to Use the Editor Mode The editor mode for result flagging is a powerful way of creating your own result flagging rules To define a result flagging rule for a selected job 1 2 3 Open the job in question in the Data context and switch to the Result Flagging tab Switch to the editor mode by clicking the Switch to Editor Mode icon Edit a rule that was created within the form mode _OR Create a new rule by clicking the Add icon amp 3 or Duplicate Selected Rule icon 3 Click the Edit button next to the Rule Label field and enter the result label for this rule Contents A 161 V7 Index The result label can be any arbitrary text or be a logic expression Expressions are built up of functions variables operators and values You can manually type in the expressions But you can also double click the items in the Functions Variables and Operators lists to insert them in the respective fields For example defining the result label as Number of peaks is NumberOfPeaks returns Number of peaks is 10 if the sample contains te
195. tofocus test chip required Measurement of stability of red laser signal Contents A318 V Index Pressure Cartridge Tests Diagnostics Test Purpose Pressure Offset Test Pressure Control Test System Leak Test Flow Cytometry Autofocus Test The vacuum system of the pressure cartridge consists of a pump and the corresponding tubes This test calibrates the pressure sensors to zero Checks if the bioanalyzer is able to hold the working pressure of 140 mbar During the test pumps stay on while the system tries to regulate pressure to be kept at 140 mbar Cell Autofocus test chip required Checks if the bioanalyzer is able to maintain a vacuum Produces a test pressure of 100 mbar and monitors for changes Cell Autofocus test chip required Checks that the optical system of the bioanalyzer is correctly calibrated Cell Autofocus test chip required NOTE With bioanalyzer model G2938A only diagnostic tests in combination with the electrode cartridge can be performed Contents A319 V Index Test Chips Depending on your bioanalyzer setup electrophoresis or flow cytometry different test chips are required to run some of the diagnostics tests Test chip kits are part of the bioanalyzer electrophoresis set G2947CA and flow cytometry set G2948CA Test Chip Kit for Electrophoresis Assays reorder no G2938 68100 Test Chip Comment Quantity Autofocus Test Chip Values for fluorescen
196. tree navigation and specify the target directories From now on chip run data is automatically exported every time a chip run has finished NOTE If you stop a chip run auto export does not take place Contents A 280 V Index Exporting Tables You can export e Result tables peak tables fragment tables and ladder tables as csv files or xls files e Log book tables as html or txt files To export a result table peak table fragment table or ladder table 1 On the Assay Properties Electropherogram Gel Histogram or Dot Plot tab right click the heading row of a table 2 From the context menu select Export The Save As dialog box appears 3 Enter a file name and choose the destination directory 4 Select csv or xls as export file format 5 Click Save TIP Result tables can be automatically exported every time a chip run has finished Refer to Exporting Chip Run Data Automatically on page 280 for details To export a log book table 1 On the Log Book tab right click a table 2 From the context menu select Export The Export Data dialog box appears Contents A281 V7 Index 3 Click the button to specify the file name the destination directory and the file type You can choose between HTML file for html output and Tabbed text file for txt output 4 Specify whether you want to export the Selected rows only or All visible rows 5 Click OK Exporting Graphs You can export graphs as individua
197. tries in the selected column in ascending order 2 Click the column header again to reverse the order To filter a log book table 1 In the Log Book toolbar click Filter T The Filter dialog box appears 2 To define a filter for events from a specified period of time specify a Start Time and an End Time 3 To define a filter for events with certain entries in a column specify the column name and the value to search for 4 Use the Filter Action radio buttons to define whether only events that match the filter criteria are displayed FilterActionHide or whether those events are highlighted while the others are still listed FilterActionHide 5 Click OK to apply the filter to the log book table The filter definition in the following example excludes all events from the Run Log in the Data context with an Event Type other than Critical Contents A 312 V Index Ix x Vv ok Ciel pas Time Stamp 7 Start Time 27 04 2005 Help End Time 27 04 2005 Show Al Vv Cancel Columns Show only O Information Source M Warning Categoy eiea Sub Category O lt Blanks gt Event Type Warning Critical AND Time Stamp 27 04 2005 27 04 2005 LA m Filter action Appropriate events only Mark inappropriate events To remove the filter from a log book table 1 In the Log Book toolbar click Reset S TIP You can hide show any of the log table columns and r
198. ts with purity 1 Specify the fragments to search for 3 Specify results The samples that contain the specified fragments Index Fragment Size bp 1 150 Shall be colored Change F ra g men t Shall be labelled Sample contains the specified fragments protein list 2 Specify options All other samples The sum of the concentrations of the specified fragments 1g percent celor Change compared to the total concentration shall be at leas Purity Tolerance For the fragment sizes search within a tolerance of 5 percent Shall be labelled fal Other Samples The specified fragment sizes should be treated such that All of them must be present Lo g i co p e rati on Any of them can be present Labels and color definitions This form can be used for searching fragment sizes along with some purity constraints 4 Define the fragment size s to be searched for 5 Define the required purity for the fragment size s and the tolerance 6 Ifyou defined several fragment sizes and want all of these to be present in the flagged samples select the option All of them must be present If you only require that one of the sizes is present select the option Any of them can be present Contents A 160 V Index Select the color with which the samples that meet the criteria should be marked Optionally select the color with which samples that do not meet the criteria should be marked Apply this rule
199. uble click the desired marker 0R Right click the corresponding row in the result table and select Configure Marker from the context menu 0R Select the marker and click the Configure Marker button in the toolbar The Configure Marker dialog box appears Configure Marker E x Name annexin Lower Value 20 34 gt 0 01 Upper Value 3438 99 lt 10000 Color eal m Cancel Help 2 Entera name for the marker for example the used dye it is advisable to use names that identify the marker 3 Enter a Lower Value left vertical line Contents A222 V Index 4 Enter an Upper Value right vertical line NOTE The lower and upper values must be within the range of 0 01 10000 relative fluorescence units 5 Click the Color button l to open the Color dialog box and select a color 6 Click OK Contents A 223 V Index How to Move the Upper and Lower Limits of Markers You can change the position of both marker lines by dragging them with the mouse 1 Position the mouse pointer on a marker line The mouse pointer changes its shape to a hand 2 Drag the line to the desired position Annexin CY5 Annexi wo pi E o gt Ww t o zZz 101 Fluorescence 3 Release the mouse button 4 Repeat these steps for the other marker line if necessary NOTE You can change the marker limits also by entering fluorescence values in the Configure Marker dialog box see How t
200. ult C 2100 bioanalyzer 2100 expert Data C Custom C 2100 bioanalyzer 2100 expert Data Tl Create daily subdirectories Cancel Help 4 Select the export categories and specify a target directory Contents A 2718 V Index NOTE Keep in mind that exporting a chip data file can require up to 20 MB of disk space In particular exporting electropherograms and gel like images as tif or bmp files may take up a lot of disk space 5 Click Export Several system dialog boxes appear one for each export category allowing you to check and modify names and locations of the export files Clicking the Save button in these dialog boxes finally starts the export TIP Chip run data can be automatically exported every time a chip run has finished Refer to Exporting Chip Run Data Automatically on page 280 for details Contents A219 V Index Exporting Chip Run Data Automatically NOTE Keep in mind that exporting a chip data file can require up to 20 MB of disk space In particular exporting electropherograms and gel like images as tif or bmp files may take up a lot of disk space To enable and configure automatic export 1 Switch to the System context and select Auto Export in the tree navigation 2 Activate the Auto Export check box 3 Specify the export categories that are to be included in the exported files for electrophoretic and flow cytometric chip runs 4 Switch to Default Export Directories in the
201. ument methods data analysis data output settings and data display settings ASY file In Bio Sizing electrophoretic assays were stored as asy files 2100 expert can import asy files See also XSY file Contents A 338 V Index Audit Trail Audit trails are available in the 2100 expert software only with the security pack installed They are used to record the activities of the logged in users and cannot be modified The audit trails as well as log books are subject to data protection Only authorized users are allowed to inspect them They are saved with the data files or into an audit file repository which is automatically archived Baseline A baseline is established just after the First Peak Time setpoint After the overall baseline is established a local baseline is calculated for each peak to compensate for baseline drift For isolated peaks the local peak baseline is simply a straight line connecting the Start Point of the peak with the End Point For peaks that are very close together an average baseline is used when the value between the peaks does not drop to the actual baseline Contents A 339 V Index The figure below shows baselines established for DNA assay peaks Peaks for DNA and protein assays are determined on a peak by peak basis the overall baseline is shown FU 35 30 25 Sample 6 tewsecces 8 loosaadaancaaa 93 leaeeoaen faeasee s lowcnecst Bhacaccucaad 62 64 66 68 70 72 74 7
202. ur own assays for example in subdirectories of the assays directory Contents A 263 V Index How to Create a Custom Assay To create a custom assay 1 2 3 eo o N 0 Switch to the Assay context From the Assays menu select an assay 0R Select File gt Open and open an assay xsy file The file appears in the Tree View Panel NOTE If you want to create a new flow cytometric assay with free gating direction or with more than one marker or region open and modify the assay Generic xsy Switch to the Assay Properties Tab to modify the assay setpoints if required Switch to the Chip Summary Tab to enter chip sample and study information For flow cytometric assays define markers and regions on the Histogram Tab Single Grid View and Dot Plot Tab Single Grid View For electrophoretic assays define flagging rules on the Result Flagging Tab Select File gt Save As to open the Save As dialog box Under Save as type select xsy and enter a name and location for the new assay Click Save to create the new assay Contents A 264 V Index How to Modify a Custom Assay NOTE You cannot save modifications to predefined assays such as Apoptosis or DNA 1000 To modify a custom assay 1 From the File menu select Open The Open dialog box appears 2 Select an assay xsy file and click Open The assay appears in the Tree View Panel and the Assay Properties Tab is displayed 3 Modify the
203. ve cells Histogram evaluation The two histograms displaying the results of the assay are related to CBNF red fluorescence and GFP blue fluorescence High fluorescence values in the red histogram indicate a staining with CBNF which is associated with living cells See the following image as example Contents A253 V Index High fluorescence value Is associated with living cells in pi c w gt w pn a O zZz Low fluorescence value indicates dead cells The values are displayed in the result table each histogram has its own table All events related to the red marker here CBNF All measured events CT Marker tase Zial Even gt HAll Events i After gating by using the red histogram in the blue histogram only CBNF stained cells are displayed High blue fluorescence values indicate GFP producing cells See the following example Contents A254 V Index GFP No of Events P no O bui v High fluorescence value indicates GFP producing cells 102 10 1 100 101 102 108 Fluorescence Amount of the CBNF containing cells in relation to all measured cells Percentage of GFP containing cells in relation to all measured cells Marker Events gt Gated by CBNF tasenees fee o E E T of gated Percentage of GFP containg cells in relation to the cells gated by the CBNF marker Contents A 255 V Index Dot plot evaluation
204. ve the upper marker do not have to be detected The peak table for the ladder well shows the peak size and concentration Lower Marker An internal standard that is added to a sample in a well to assist in determining size of the sample The lower marker is the same as the first peak found in the DNA ladder M Method Methods are available in the 2100 expert software only with the security pack installed A method is referred to as an electrophoretic or flow cytometric assay with additional information stored to it This additional information includes instrument information study information report settings and workflow definitions Microfluidics The movement of liquids through micro fabricated structures by means of electrical fields or pressure vacuum holding the promise of greater functionality with significantly improved reliability e small glass or plastic devices with micro channels as experimental platform e active control of fluids without moving parts on chip through miniature electrodes or pumps controlled by software scripts e emulation of conventional liquid pumps valves dispensers reactors separation systems etc Contents A 351 V7 Index e capability of liquid transfer separation dilution reactions and more Molarity Concentration 10 nmol where Molarity is measured in nanomoles per liter nmol l Concentration is measured in nanograms per microliter ng pL Size is measured in base pairs
205. y files in the 2100 expert installation folder under diagnosis If tests fail send the xdy files to the Agilent service Contents A324 V Index Performing Verifications To ensure a validated Agilent 2100 bioanalyzer system verification steps have to be performed at installation and operation level 2100 expert allows for detailed installation verification and system verification on both the bioanalyzer hardware and software Each verification comprises a series of tests and measurements that you can run and document in the Verification context of the 2100 expert software Installation Verification Installation verification includes tests to verify that the bioanalyzer software and hardware are installed properly and that all electrical and pressure connections are correct Installation verification must be performed once after installation Contents A 325 V Index System Verification System verification proves that the bioanalyzer system is suitable for its intended use that is that it will function according to its operational specifications in the selected environment System verification should be performed e at first use of the instrument e after relocating the instrument e after changing essential parts of the system for example software updates or exchange of cartridges e after instrument repair e on regular time intervals Verification Procedure To perform verification tests 1 Switch to
206. yzing and Evaluating the Results of a Flow Cytometric Assay on page 211 Print the results to document them on paper or an electronic format such as HTML or PDF See Printing Reports on page 286 Export the results or parts of them for further evaluation in other applications See Exporting Data on page 277 Compare the results with the results of other chip runs in the Comparison context See Comparing Samples from Different Electrophoretic Chip Runs on page 143 Insert the next chip in the bioanalyzer and start a new chip run Contents A21V Index Looking at 2100 Expert Before you start running assays on the Agilent 2100 bioanalyzer you should familiarize yourself with the 2100 expert software e Introduction to the Key Features of the 2100 expert on page 29 e Starting 2100 Expert on page 31 e 2100 Expert Work Area on page 32 e Closing 2100 Expert on page 43 Contents A28V Index Introduction to the Key Features of the 2100 expert The Agilent 2100 expert is characterized by the following key features 2100 expert provides a single software platform with a common user interface for running analyzing evaluating presenting and comparing DNA RNA protein and cell parameters 2100 expert is installed in one go After installation the functionality for electrophoretic and flow cytometric assays can be activated separately with license keys 2100 expert provides an optional se

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