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User`s Guide for IonWorks® Quattro

1. PLATE 2 BUFFER Figure 2 Diagram of the experiment deck Removable Reservoir Three types of removable reservoirs can be used on the experiment deck V bottom Flat bottom Multi well These are used as follows The 5 mL V bottom reservoir is used in either the CELLS or the BUFFER positions The 10 mL flat bottom reservoir must be used in the SOAK position 12 or 48 reservoir multi well boats for use with the 12 and 48 channel pipettors respectively can be used in either the CELLS or the BUFFER positions 17 User s Guide for lonWorks Quattro 18 Caution Placing the V shaped reservoir in the SOAK position will cause damage to the E Head Using a 12 well boat in an instrument configured with a 48 channel pipettor or using a 48 well boat on an instrument configured with a 12 channel pipettor will cause damage to the pipettor Ensure that the reservoirs are positioned in appropriate stations and are loaded with the proper solutions prior the start of an experiment After the experiment is completed discard unused solutions and rinse the reservoirs with a 50 ethanol solution followed by DI water 2 3 2 Electronics Head Soak Station The soak station houses a removable flat bottomed reservoir for soaking all 48 probes of the electronics head simultaneously The station is used to apply
2. 89 User s Guide for lonWorks Quattro 4 4 2 Plate The Plate section at the top left of the data file window shows a diagram of either the PatchPlate or the compound plate change views with the left most button in the section s title bar Wells are color coded according to their having met or failed threshold values for the hole and seal tests or other user configured criteria set in the Filters and Summary sections below The Summary section settings at any time use a particular metric definition as well as the data i e the scan or scans these are applied to The individual metric values for each well are displayed in a tooltip when the cursor is held over the well Yellow colored cells to represent hits show whether the metric for a cell meets a threshold also set in the Summary section Other colors indicate wells removed from analysis for failing to meet basic criteria of well or cell health For compound plate wells four values from the selected metric are combined from the four replicate wells on the PatchPlate The value displayed is either the average minimum or maximum of the group as set in the Summary section The numerical value is shown in the tooltips and used for the color coding Information boxes within the Plate section present statistics for the entire plate at the current settings See Plate Legend and Statistics Area below for details of these Double clicking on a well opens a new windo
3. Save Active Protocol Saves the currently active protocol as an ihp file Save Active Protocol As Saves a copy of the current protocol with a new name to a user selected location Export Traces Opens the Configure Trace Export dialog to select raw trace data to export Traces are exported in ASCII text file format with ihd file extension See Configure Trace Export Dialog page 100 Export Metrics Opens the Configure Metric Export dialog to export analysis results from the metrics defined in the experiment The results are exported as ASCII text files iha See Configure Metric Export Dialog page 101 The most recently opened data and protocol files 1 2 3 up to 10 Click on a file to reopen it data files or load it protocol files Closes the program application If you have Exit unsaved data you will be prompted to save it before closing 43 User s Guide for lonWorks Quattro 44 4 2 2 Edit Menu A description of the Edit menu items is listed below Item Description Active Protocol Opens the Protocol Editor dialog to edit the experimental parameters of the currently loaded protocol reported in the Status Bar User Metrics List Opens the User Metric List Editor dialog to create edit or delete analysis measurement metrics for the selected data file Metrics can also be imported from other data and protocol files See Analyze Export Da
4. Trace Groups sssssssssssisessiessees 46 User Metric Editor 81 Utilities Menu 49 Vacuum Requirements 6 Voltage Protocol Editor 4 57 58 59 64 83 84 2 23 Voltage Clamp Voltage Offsets Window Menu 51
5. button to the right and remove by clicking the button Click on an interval button to open the Interval Settings dialog for that interval Interval Settings This dialog is opened by clicking on an interval button in the Interval Sequence section Use it to configure the voltage and duration of the interval Interval Duration The duration of the interval up to 5000 milliseconds Starting Potential The voltage stepped to at the start of the interval range 1000 mV to 1000 mV Ending Potential The voltage at the end of the interval If the same as the Starting Potential then the single voltage is maintained for the duration of the interval a voltage step If different from the Starting Potential then the voltage increases or decreases regularly throughout the interval to deliver a voltage ramp Enable CV Family Sets the command voltage to be repeated and the voltage in this particular System Software Item Description interval to increase or decrease a set amount with each repetition Only available for voltage step intervals where the staring and ending potentials are the same Enabling a command voltage family disables conditioning trains and command voltage sequence repetitions Step Potential by Sets the number of millivolts to increase positive value or decrease negative value the interval with each repetition of the command voltage Number of Steps Sets the number of
6. If a boat type plate is used it must be added to the plates txt file and properly calibrated The pipettor will move to different positions within a boat type plate Wash Cycles Select the number of standard wash cycles up to 20 used to wash the pipettor after each compound transfer sequence Pipettor Transfer Options Compound Mix Cycles Select the number of compound mix cycles from 0 to 10 to mix compound before aspirating compound for delivery to the PatchPlate 69 User s Guide for lonWorks Quattro 70 Item Description Use Extra Aspirate Check and enter an additional volume of compound to aspirate to give highest pipetting performance Setting extra aspirate marginally increases experiment time Use Singleton Dispense Check to aspirate each compound delivery to the PatchPlate independently rather than aspirating sufficient compound in a single draw for four wells This improves the dispensing precision for the four replicate wells This option is only enabled for the 12 channel pipettor when dispensing quadruplicates Note f Singleton Dispense is used with Use Extra Aspirate the extra aspirate volume is drawn with each transfer not divided among all four singleton transfers Singleton Dispense adds roughly 2 5 minutes to the total running time of a PatchPlate shown in the Measurement Timing vs Scan Type table Pipettor Prewetting Option Enable for use with sticky com
7. Print Prints the graph currently on display in the editor When Sequence Repetitions are enabled for either the command voltage or the conditioning train this command toggles between viewing the Show Hide Repetitions repeated intervals or not Repetitions are always shown when Scan Sequence is selected below When Scan Sequence is selected toggle between the waveform voltage as delivered to one 12 head read bank or the sequence as delivered to all four read banks All Single Read Banks Ensure that the graph is showing your latest Refresh Plot d configuration changes 59 User s Guide for lonWorks Quattro 60 Command Voltage Set the sampling rate and pre signal pulse in this view of the dialog To define the command voltage click the button in the right of the Interval Sequence section to add intervals then click on these to open a subdialog where you configure it Press the button to remove a selected interval Use the Sequence Repetitions section in the bottom right of the dialog to repeat a contiguous sequence of intervals within the command voltage Item Description Interval Sequence Command Voltage Protocol The number of intervals within the command voltage is represented on the left of this section by the number of buttons Interval buttons are numbered left to right except for the family interval if configured Add intervals by clicking the
8. cccccccsssssssesesesesesesesesesesesesssssssesesesscsssssesessseseseeeees 18 2 3 3 Electronics Head Wash Station c cccccccssssssssesesessssessesesesesssssssssssecscscsessssssesssesenes 18 2 3 4 PatchPlate Station with Clamp Frame cccccccessetesesesssnenesescseeeeeeceneneeseseseseenenenes 18 2 3 9 Pipettot Wash Statloniic scseivscdcevstinecuctesneves eiae sean e i iaaa 19 2 3 6 COMPOUNG Plates yiera ri i eo ee en EAO eA E Ea EAE T eetoeenses 19 23 7 Cell SUSPENSION RESET VOIE sss ceisiecenecssssssucseh heeds ieSesea esta teea tu tu i a a a 20 23 9 SOLVENE BOE esise cysdvaiestubeg E AEEA AE e E A NES 20 23 9 B ffer RESELV OIE essence ire oaei i R RE EEE NER 21 24 1270r 48 Changel Pipetto kinners eerie sivas oan eiieeii nin 21 2 4 1 Pipettor Mechanism of Action ccccscsssssesesceeseeseneesesesesnsnsnesesessseeseenssessseeseeseenenenes 21 2 0 48 Channiel Electronics Head isisisi aea Eea EEEE E EE E a EE a ea 22 2 5 1 Electronics Head Mechanism of Action The Amplifier System ccceee 22 2 6 PatchPlate Consumable esis ieren iiaea en a eabsnesbeossta ipseenchossgsbendseedensedacheate 23 2 6 1 Well Arrangement and Capacity ccccscsesessecesesetsteesesesssnenenesssesneneenessssesesseeeeenenes 24 2 6 2 PatchPlate Packaging va csie crest ioen elite ih Aine ove eared e ae 25 2 7 Electronics CADINEL s ossessi orien iiie nr e A RT Ea E E SEE AA STRE aR 25 271 Power Supply MO ule serprent eirsrieen an aie peera e ei a a
9. Median of the summary values across the PatchPlate including or excluding filtered out results depending on Apply to Filtered Data setting Standard deviation of the summary values Minimum summary value Maximum summary value If you highlight a row the name of the summary it reports appears in the Statistics title bar Alternatively hold the cursor over a row and the summary name is reported in a tooltip Summary rows in the display can be reordered using buttons in the section title bar The title bar also has a button to copy the entire contents of the display to the clipboard 99 User s Guide for lonWorks Quattro 100 4 5 Data Export The IonWorks software allows export of metrics analysis data and raw trace data to be configured in protocols so that the data is exported automatically whenever the protocol is run Data is exported in ASCII tab delimited text files The same export options are available for saved data files In addition images of selected trace data can be exported for individual wells or groups of wells The different export options are described in the sections below 4 5 1 Configure Trace Export Dialog Configure the export of trace data with the Configure Trace Export dialog Trace data is exported as tab delimited ASCII text file with ihd extension A separate file is written for each well You can configure automatic export of trace data within the protocol definition Au
10. System Software Item Description give 2x or 3x dilution as shown in the table the default setting is bolded Compound Buffer Cells Dilution JO 3 0 3 0 3 0 3x 9 0 3 5 3 5 3 5 3x 10 5 4 0 2 0 2 0 2x 8 0 5 0 2 5 2 5 2x 10 0 Buffer and cell volumes are reported at the bottom of the section in the dialog Select Timing and Scan Parameters Compound Incubation Time Enter the length of time that you want the cells incubated with compound before applying the voltage stimulus value range 1 s to 1200 s Note that the incubation time does not start until after the addition and mixing of the compound The Minimum Incubation Time includes the mixing time and the Maximum Incubation Time includes mixing time and in some cases washing time depending on the scan mode Number of Scans Before Addition Set the number of scans in the pre compound set scans Number of Scans After Addition Set the number of scans in the post compound set of scans Delay between Scans Set the time between scans within each of the pre and post compound sets of scans This time does not apply to the duration between the two sets of scans when compound is applied Total experiment time and the final file size are reported at the bottom of this section Solvent Wash Options Check to enable a solvent wash after each row of compounds are dispensed and mixed in the PatchPlate The pipettor washes
11. Absolute Value of Mean The absolute value of the mean 83 User s Guide for lonWorks Quattro 84 Item Description After choosing the reduction choose a time interval within which to measure the reduction Entire Signal Find the selected reduction value from the entire duration of the command voltage Window From To Set a time range in milliseconds within the command voltage to calculate the selected reduction You can set From and To values by using the cursor and popup menu in the graph at the top of the dialog or enter values directly Combine Reduction If you chose a Compound metric type select a method of combining the two reductions The order that the reductions are combined is set by the combination method so if you want to reverse the order you must reconfigure e swap the reductions The following combinations are available Absolute Difference Absolute value of Reduction 1 Reduction 2 Absolute Difference Absolute value of 100 x Reduction 2 Reduction 1 Reduction 1 Ratio Reduction 1 Reduction 2 Absolute Value of Ratio Absolute value of Reduction 1 Reduction 2 Difference Reduction 1 Reduction 2 Difference 100 x Reduction 2 Reduction 1 Reduction 1 Family Scans If a command voltage family is configured in the Voltage Protocol Editor each member trace of the family will produce a value in the ex
12. Use only with adequate ventilation User s Guide for lonWorks Quattro Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended on the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal 1 3 2 Chemical Waste Hazard Warning WARNING CHEMICAL WASTE HAZARD Wastes produced by Molecular Devices instruments are potentially hazardous and can cause injury or illness Read and understand the material safety data sheets MSDSs provided by the manufacturers of the chemicals in a waste container before you store handle or dispose of chemical waste Handle chemical wastes in a fume hood Minimize contact with and inhalation of chemical waste Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or clothing 1 3 3 About MSDSs Some of the chemicals used with this instrument may be listed as hazardous by their manufacturer When hazards exist warnings are prominently displayed on the labels of all chemicals Chemical manufacturers supply a current MSDS before or with shipments of hazardous chemicals to new customers and with the first shipment of a hazardous chemical after an MSDS update MSDSs provide you with the safety information you need to store handle transport and dispose of the chemicals safely We stron
13. at current settings Times are affected by the Compound Incubation Time Scan Type and Use Extra Aspirate Use Singleton and Pipettor Prewetting pipettor options System Software Item Description Tip Refer to the Minimum Incubation Time in the summary table and use this value for the Compound Incubation Time in order to avoid being prompted with a warning message For scan modes other than EAO the Compound Incubation Time must be sufficiently long to allow one dispensing mixing and washing sequence of the pipettor otherwise a warning appears and the incubation time is automatically reset Check to enable a solvent wash after each row of compounds are dispensed and mixed in the PatchPlate The pipettor washes at a different row of the solvent plate after each row of the compound plate After washing in solvent the pipettor is washed as usual in the pipettor wash station The solvent wash plate should have sufficient volume in the wells to exceed the dispensed compound volume Wash Cycles Specify the number of solvent wash cycles up to 20 Each cycle consists of one aspirate and one dispense of solvent in and out of the Solvent Wash Options __ pipettor Location Select the location of the solvent You can use a plate location if solvent is in a standard size plate or the solvent boat Note The plate location is automatically set to be the location other than the location selected for compound
14. 6 3 Detecting a Salt Bridge Sometimes salts can accumulate on the electrodes forming a bridge and causing a short across the pins on the E Head This test is designed to detect this problem To detect a salt bridge Step Action 1 Launch the lonWorks software OJOJ AJOJN Fill the soak boat with 10 mL of Ca Mg free PBS Select Soak Electrodes from the Utilities menu Enter 300 seconds when prompted Upon completion of the utility execution select Test Electrodes Click Acquire If there is no salt bridge all pins should read N A Any other value indicates a short across the pins 135 User s Guide for lonWorks Quattro 136 6 4 Testing the Pipettor The Test Pipettor utility allows you to test the dispensing accuracy and precision of the pipettor The utility will use the lonWorks Pipettor to transfer fluid from a source plate located in plate position 2 to a test plate located in plate position 1 A standard gravimetric test is recommended to measure accuracy while an absorbance or fluorescence test is recommended to measure precision How to use the test pipettor utility for an absorbance precision test Step Action 12 Channel Pipettor 48 Channel Pipettor Prepare a 0 8 mM tartrazine solution in 100 mL DI water 30 mL required per run Pipette 300 mL per well into a 96 well Pipette 80 mL per well into a source plate Place into the Plate 2 384 well source plate
15. Addition Scan Parameters dialog The first option in the dialog sets repeated scans The other two options allow the command voltage to be edited between scans of the whole or part of the PatchPlate useful during assay development The Family Mode option must be used if there is a family configured in the command voltage otherwise use Interactive Mode Item Description Run a set number of scans over the PatchPlate area configured for use at Prime Plate Repetitive Mode Set the number of seconds between each of the scans Run the scan over the PatchPlate area configured for use at Prime Plate After a scan you have the option to reconfigure the command voltage and then scan again Interactive Mode This option is not available if you have a family configured in the command voltage Scan plate by EAO Mode Scans just an eighth of the PatchPlate before giving you the option to edit 65 User s Guide for lonWorks Quattro 66 the command voltage and either rescan the same eighth scan the next eighth or terminate the experiment For use when you have a family configured in the command voltage this option scans an eighth of the PatchPlate then offers you the chance to edit the command waveform Family Mode Note Subsequent scans always move to the next eighth you cannot rescan an eighth as in Interactive Mode The number of members in the command voltage family cannot be changed while in
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17. Menu Bar has eight menus reduced to six when no data files are open The menus and their main features are File General file opening closing and saving and file export Edit Protocol experimental metrics and results filter editing View Interface options and protocol and experiment summaries Experiment Instrument control to run experiments Utilities Instrument control for specific maintenance operations Data Filter and correction options for raw data traces in data files Window Display options for data files Help Includes PDF file of this manual Descriptions of all commands are given below Commands requiring further explanation have dedicated sections further in the chapter 4 2 1 File Menu Commands in the File menu are listed below Some commands are not available when no data file is open 42 System Software Item Description Open Data File Opens a previously saved data ihe file Close Data File Closes the current data file Save Data File Saves the currently active data file Save Data File As Saves a copy of the current data file with a new name to a user selected location New Protocol Opens the Protocol Editor dialog to configure a new protocol The new protocol is automatically loaded identified default in the Status Bar Open Protocol Opens a protocol from either a previously saved protocol ihp or data file
18. Right click on the chart and select Export Dialog form the menu In the Export pane select the desired export format In the Export Destination pane select the desired output destination 5 In the Object Size pane specify the size of the file if outputting it as a picture When you have selected all the appropriate options click Export 6 If you select File as the Export Destination click Browse and navigate to the desired directory for saving the file 5 11 2 Exporting Data for Multiple Wells To export the data for multiple wells in tab delimited format Step Action 1 Open the data file from which you want to export Select Export Traces from the File menu This opens the Configure Trace Export dialog box 124 Operating the System Step Action In the Export By and Wells to Export panes select the wells from which you want to export the data Note Wells exported when using the Along Row option will be different than for the Along Column option if the selected wells span more than one row or column The exported wells wrap around to the next row or column as shown in the following example Along Row from a3 to a16 Files a3 a4 a5 a6 a7 a8 a9 a10 a11 a12 a13 a14 a15 a16 Along Column from a3 to a6 Files a3 63 c3 d3 e3 f3 93 h3 a4 b4 c4 d4 e4 f4 94 h4 a5 65 c5 d5 e5 f5 95 h5 a6 a16 In the Data Sets to Export select the data reads you want to export To 4 sel
19. When the run is completed the Trace Groups window will close and the Data file displaying the results of the run will appear in the main window For a description of the Data File window see Data Files page 86 5 10 8 Post Experiment Operations Wait for the experiment run to complete all its operations The following indicates completion of all operations The experiment timer will finish its count down Operating the System The primary analysis window will display the PatchPlate view along with a summary of the experiment data The traffic light icon on the main window toolbar of the system software will turn green indicating the experiment is completed and the software is ready for the next run When the experiment is completed Step Action 1 Open the instrument access door 2 Remove the v bottom removable reservoirs from the Cells and Buffer stations on the experiment deck Discard the contents and clean the removable reservoirs D Important If reusing the v bottom reservoirs should be cleaned thoroughly with 50 EtOH and then DI water so that the risk of contaminating the next experimental run is minimized If necessary remove the compound plates from the experiment deck Important Compounds will adsorb to plastic over time and therefore we do not recommend re using plastic compound plates for subsequent experiments Compound adsorption is greater for hydrophobic compou
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21. and the reference scan values which are the same but the post compound metrics will have interesting results This case would use the Cross Scan Reduction option to produce a comparative value across multiple scans relative to the selected reference scan One value will still be output per scan Note If a metric definition spans a family it cannot be configured to combine values taken before and after compound addition This case would require two Cross Scan Reductions in combination and is not supported Item Description Use this graph to assist in identifying time or voltage settings for analysis metrics The x and y coordinates of the cursor are reported in the field just below the graph Zoom To zoom into a region drag the cursor over that region To undo zoom use the popup menu option Command Voltage Time Tag Click in the graph to add a labeled Graph vertical marker to use as a time reference Click elsewhere in the graph to replace this with a tag in the new position Set From To When the Window From and To option is selected below to define a measurement region position the cursor at the start and end of the region and use the popup menu Set From and Set To commands respectively to populate the fields Enter or edit a name for the current metric in the field i Simple Take a single measurement reduction Define Type from a defined region of the command voltage Metric Name Sys
22. and or pipettor Fill the Soak reservoir with 10 mL of chloride solution i e bleach O Important Do not fill the flat bottom reservoir in the Soak station 5 with less than 9 8 mL or more than 10 mL of solution Under filling can cause inadequate soaking height of the probes on the E head Over filling can immerse and wet the clamping bar that holds the E head probes and will cause an electrical short circuit between the probes and Maintenance Step Action E head Close the instrument access door and turn the handle to the lock position Wait for the traffic lignht button on the main window toolbar to turn green and then select Rechloride Electrodes from the Utilities menu Choose the number of repeat cycles and soak or dip durations If a dip option is checked the dip period duration duty cycle and total number of dips can be specified Click OK to start rechloriding the probes The E head will move to the soak station and immerse the 48 probes into the reservoir for the specified duration The E head is then rinsed at the E head wash station After the chloriding and wash steps are completed the E head will return to its home position Wait for the traffic lignht button on the main window toolbar to turn green and then open the instrument access door and remove the flat bottom reservoir from the Soak station 10 Discard the contents of the Soak reservoir and wash with distilled water
23. below Note This menu is only available when a data file is open Command Description New Window Opens another window on the currently selected data file Cascade Arranges the currently open windows in a cascade pattern Tile Arranges the currently open windows by tiling the main window Arrange Icons Arranges the minimized windows 1 10 data files Selects an open data file window and brings it to the foreground 4 2 8 Help Menu A description of the Help menu items is listed below Item Description About lonWorks v2 Opens a dialog box that displays the version of the lonWorks Quattro system operating software and the version of the firmware currently running on the instrument User Manual Opens a PDF version of the latest lonWorks Quattro System User Manual See also What s New below 51 User s Guide for lonWorks Quattro Item Description Opens PDF files with compound delivery maps for the 12 and 48 channel pipettor and includes examples of full and partial plate coverage scenarios Compound Plate Maps Opens a PDF describing the new features introduced in the current version of lonWorks operating software and may not be fully documented in the User Manual as yet What s New 4 3 Protocols The IonWorks software controls experiments on the lonWorks instrument with a set of instructions called a protocol Only one protocol calle
24. below describes the contents and properties of these source bottles Source Bottle Capacity L Dead Volume mL Contents F Head Wash 2 100 Pipettor wash solution E Head Wash 1 50 E Head wash solution Internal Buffer 2 100 Internal buffer solution Antibiotic 1 50 Perforation agent in internal solution F Head wash E Head wash Internal buffer Antibiotic Figure 4 Source bottles in rack Each bottle is mated to a labeled cap The caps contain filling ports with barbed fittings and are connected to tubing lines that provide a continuous path to the appropriate station on the experiment deck Tube lines terminate at the waste bottles 29 User s Guide for IonWorks Quattro 30 Note Source bottles must be housed at the correct positions on the holding rack 2 9 2 Waste Bottles Two 4 L waste bottles are housed on the holding rack at the right side of the system cabinet The vacuum waste bottle collects fluid waste from the wash stations on the experiment deck The plenum waste bottle collects fluid waste from the PatchPlate station Each bottle is mated to a threaded cap that contains filling ports The filling ports are connected to tubing lines that transport the waste fluids from the various stations to the appropriate source bottle One of the filling ports on the vacuum waste bottle cap serves as the entry point for the systems main vacuum line The waste bottle
25. dialog for that interval System Software Item Description Interval Settings This dialog is opened by clicking on an interval button in the Interval Sequence section Use it to configure the voltage and duration of the interval Interval Duration The duration of the interval up to 5000 milliseconds Starting Potential The voltage stepped to at the start of the interval range 1000 mV to 1000 mV Ending Potential The voltage at the end of the interval If the same as the Starting Potential then the single voltage is maintained for the duration of the interval a voltage step If different from the Starting Potential then the voltage increases or decreases regularly throughout the interval to deliver a voltage ramp Enable Conditioning Trains Check to include a conditioning train in your waveform Note Conditioning trains cannot be included if you have a command voltage family Holding Potential Set a holding potential to follow the conditioning train until the command voltage starts This holding level is maintained for the same duration as the pre signal interval Apply Trains Select to run the conditioning train with scans before and after compound addition or only with the scan that follows compound addition Skip Trains for 1 Pre and Post Scan RSA Experiment Only If the selected experiment type is Repetitive Scans with Addition this option will not apply the Condition
26. enable a solvent wash after each row of compounds are dispensed and mixed in the PatchPlate After washing in solvent the pipettor is washed as usual in the pipettor wash station Each of the four basins should have approx 4 mL of solvent Wash Cycles Specify the number of solvent wash cycles up to 20 Each cycle consists of one aspirate and one dispense of solvent in and out of the pipettor 73 User s Guide for lonWorks Quattro 74 Item Description Location When dual compound additions are selected solvent can only be located in the solvent boat Pipettor Transfer Options Wash Cycles Select the number of standard wash cycles up to 20 used to wash the pipettor after each compound transfer sequence 1 amp 2 Compound Mix Cycles Select the number of compound mix cycles from 0 to 10 to mix compound before aspirating compound for delivery to the PatchPlate Use Exira Aspirate Check and enter an additional volume of compound to aspirate to give highest pipetting performance Setting extra aspirate marginally increases experiment time Use Singleton Dispense Check to aspirate each compound delivery to the PatchPlate independently rather than aspirating sufficient compound in a single draw for four wells This improves the dispensing precision for the four replicate wells This option is only enabled for the 12 channel pipettor when dispensing quadruplicates Note If Singleton Di
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28. fluid flow These valves also perform a pulsing sequence during the priming operations to clear bubbles from the system Housed at either side of the system cabinet input and output bottles provide the source and the waste containers for the fluidics system One of the output bottles provides the entry point for the system s main vacuum line The vacuum line is routed to the following locations PatchPlate plenum Vacuum regulator Pipettor wash station Electronics head wash station 2 8 2 Plenum and Debubblers The plenum provides a vacuum sealed receptacle for holding the PatchPlate and for performing plate operations such as priming debubbling and exchanging fluids A clamp frame along with a vacuum entry orifice on the plenum s top surface is used to seal the PatchPlate A pressure gauge on the fluidics 27 User s Guide for lonWorks Quattro system monitors the PatchPlate plenum seal and reports the vacuum pressure in mm Hg The vacuum sealed lower chamber that is created at the underside of the PatchPlate allows for various priming and fluid exchange operations The plenum contains input and output ports that are connected to source and waste bottles respectively A tubing network with inline peristaltic pump and pinch valves feed the input port of the plenum Appropriate solution can be circulated through tubing lines to prime and to remove any air bubbles Two integrated vessels provide inline traps for r
29. handle to the lock position When the experiment start button is activated indicated by a green traffic light on the main window toolbar select Flush and Rinse from the Utilities menu The system will perform a flush and rinse cycle to clean the fluid lines and 12 channel pipettor When the cycle is completed the start button will turn green 117 User s Guide for lonWorks Quattro Step Action 6 After completion of the flush and rinse cycle open the access door discard the contents of the v and flat bottom reservoirs clean with 50 EtOH and DI water and reinstall for subsequent runs 5 9 Setting Up the Experiment Protocol 5 9 1 Loading and Modifying the Default Protocol To set up the experimental protocol Step Action In the main window of the software select Active Protocol located under the View menu or press the Edit Active Protocol button on the a gt E toolbar Check the experiment settings in the Active Protocol Summary window If you need to change a setting open the protocol editor from the Edit menu in the summary window When the protocol settings are correct close the Active Protocol Summary window and start the experiment It is advisable to save the protocol and give it a unique name before starting the experiment Note Fora description of protocol options and settings see Protocols page 52 5 10 Completing the Experiment Setup 5 10 1
30. more or less than two additional scans For the Single Compound Addition there will be two scans labeled Pre compound and Post compound but if the Interactive Scan mode is used there can be one or more scans labeled Scan 1 etc Define Data This section defines the single metric per well for which you will set a threshold to define the filter Metric Select a predefined metric to be used It will be applied to the scan or scans selected in Define Data Define new metrics Edit User Metric List if the metric you want is not present Define Screen y P Cross comparison The mathematical relationship used to compare the metric values from Scan A and Scan B The default choices are Difference 100 Scan B Scan A Scan A Absolute Value of Difference Difference Scan B Scan A 94 System Software Item Description Absolute Value of Difference Ratio Scan B Scan A Absolute Value of Ratio Define the filter threshold Metric values that fall within the range defined in this section are filtered out Discriminator Select Less than or equal to or Greater than Benne Thresnoid Threshold Value The threshold metric value Note When determining a threshold value for use with the filter be aware that the values displayed in the Plate Legend and Statistics Area are rounded to three decimal places whereas the values used by the Hits Display are much higher precision Values
31. one well To view the data from multiple wells hold the CTRL key down while selecting the desired files 4 When finished selecting the files you wish to open click Open This will launch the three step File Import Wizard 5 Accept the default settings by clicking Next and Finish 6 Each data set will appear as a pair of columns of time and current or voltage 5 13 Exporting Metrics When exporting the metrics from an experiment the values obtained for all wells in the plate for each metric defined in the experiment protocol is exported If the automatic data export function is enabled then the metric results file would already be exported to your designated file directory see Export Analyze Data Tab on page 79 for more information about this option If the automatic data export function is NOT enabled and you want to export the metric results use the following procedure Operating the System To export the metrics from an experiment run Step Action 1 From the File menu in the main window select Export Metrics The Configure Metric Export dialog box opens Choose the desired options see Configure Metrics Export Dialog on page 101 for information on the options in this dialog box Click OK The Save As dialog box opens In the Save in field navigate to the directory folder for which you want the file saved and type a name for the file in the File Name field Click Save to save the met
32. scans when two scans are selected Threshold value used to define hits As well as determining the contents of the Plate display the scans selected determine the traces displayed in the trace display area Configuration of a Summary analysis is essentially the same as configuration of a filter described in the section above Any number of new Summary definitions can be added with the Add button Once defined select which of the summaries to view by scrolling through the Number field or click in the row for that summary in the Statistics section below Item Description Select the summary number When a number is selected The fields in the Summary section are populated Number with the settings for that summary The Plate display shows the summary results Traces in the Trace Display show the scan or scans defined in the summary System Software Item Description Name The name of the current summary For a new summary created with the Add button the name comes up as Summary n delete this and type in a name for your new summary Add Press to add a new summary The new summary has default name Summary n where n equals the summary Number Remaining fields in the section similarly take default settings Remove Deletes the currently selected summary Apply to Filtered Data Check to exclude filtered data from the summary statistics When unchecked fi
33. should provide a final cell suspension with isolated single cells The preparation methods for disassociating cells should neither harm the cell membrane nor affect the ion channel expression The gentle mixing action of a standard hand pipettor e g 200 uL capacity has proved the preferred method for triturating cells The number of aspirating and dispensing cycles should be optimized for each cell line along with the speed and forcefulness of the trituration so as to mix the cell suspension gently 108 Operating the System 5 3 3 Window of Opportunity The cell clock starts at the instant when cells are removed from their growth media According to this clock the window of opportunity for recording stable electrophysiological response is limited Within this time frame all preparation steps must be planned scheduled and executed to optimize the experiment performance Most preparation techniques require spinning cells in a centrifuge for removing cell growth media It is recommended to centrifuge the cells at 50 x g for 4 min 5 4 Preparing Buffer Solutions The balanced salt solutions are used to maintain cells for a short time in a viable condition rather than to promote their growth During this time the cells need to be maintained in a medium of physiological pH and osmotic balance The choice and formulation of these buffer solutions depend on the cell line and the ion channels of interest The lonWorks Quattro system
34. shown in the Plate pane Statistics Reports key parameters for each of the Summary definitions The window layout is illustrated in Figure 7 System Software File Title Bar Plate Filters Trace Display Summary Statistics Metric Summary Figure 7 Diagram of Data File Window 4 4 1 Trace Display Area The right hand side of the data file window displays traces from the well selected in the Plate display on the left For a PatchPlate well traces from the selected well are shown in one graph For a 96 well compound plate four graphs show the traces from the four PatchPlate wells that received compound from the selected compound plate well The PatchPlate well location is reported above each graph The trace types shown in the graph pre compound or post compound scans or the hole or seal tests are set by the scans selected in the Define Data group in the currently selected summary in the Summary section to the left Below the graph the Metric Summary table displays metric values for the selected well or wells Values are given for each of the scans run on the well one metric at a time Select the metric from the list box immediately 87 User s Guide for lonWorks Quattro 88 above the table The time that each scan was run on the well is also reported Toolbuttons at the top of the display area offer scaling options and you can use these to view the command vo
35. this mode Liquid Junction Enter an offset correction value in millivolts to Potential Correction subtract during scans 4 3 7 Scan Parameters Single Compound Addition To configure scans with one compound addition select Single Compound Addition in the experiment type options in the Add Compound Measure Currents section and then press Scan Parameters Editor This opens the Single Addition Parameters dialog A graphic at the top of the dialog helps identify plate locations in the IonWorks Quattro In the fields below select plates for compound and solvent and configure compound additions mixing and washes You can prewet pipettor tips to help avoid losing sticky compound and set voltage offset correction Importantly set how much of the PatchPlate to deliver compound to before scanning you can apply compound to the entire PatchPlate and then proceed to scan it or apply compound to sections and scan after each Smaller sections mean cells spend less time on average in compound before scanning however at the cost of longer overall experiment time System Software Item Description Select Plate Types Select a plate type for the compound plate and for the solvent plate if using enable a solvent plate at Solvent Wash Options section Set which plate 1 or 2 is the compound plate in the Compound Location field in the next section The Plate Type list has predefined compound plates used with th
36. to 50 mL of Internal buffer e g 50 mL centrifuge tube and vortex for 1 min If using the Antibiotic source bottle the minimum working volume is 100 mL Note Amphotericin is light sensitive and will degrade over time We recommend preparing amphotericin fresh for each run Fill the Internal Buffer source bottle with internal i e pipette buffer Note Each experimental run uses 100 mL of the internal solution from the Internal Buffer bottle Adjust the final volume used in the Internal Buffer bottle based on the total number of intended runs Remember to account for the dead volume in each bottle Fill the Electrode Wash and Pipettor Wash source bottles with appropriate wash solution The recommended wash solution is Ca Mg free External Buffer of choice or Ca Mg Free Dulbecco s Phosphate Buffered Saline 1X solution Note Each experiment run uses 100 mL of the wash solution in each wash bottle Adjust the final volume of the wash bottles based on the total number of intended experimental runs Remember to account for the dead volume in each bottle Load all source bottles Note Check that the label on the bottle can matches the labelina on the 115 User s Guide for lonWorks Quattro 116 Step Action appropriate source bottle The bottle cap contains the line that connects to the appropriate station The bottle cap is distinguished by a label that corresponds to its matching
37. 2 2 System Cabinet The main system cabinet has two main compartments upper and lower Experiments occur in the upper top opening compartment This compartment houses Experiment deck Integrated 12 or 48 channel pipettor 48 channel electronics head Upper debubbler The lower compartment houses 15 User s Guide for lonWorks Quattro Fluidics system Electronics Access the lower compartment through the front opening door on the left of the cabinet Note Opening the front access door terminates any system operation Racks to hold the source and waste fluid carboy bottles are located at the left and right ends of the cabinet The cabinet is supplied with casters and leveling feet for facilitating instrument transport and for securing the system in a stationary position 2 3 Experiment Deck 2 3 1 Components of the Experiment Deck The experiment deck has the following components Electronics head soak station Electronics head wash station PatchPlate station with clamp frame Pipettor wash station Compound plate 1 Cells suspension reservoir Solvent boat Compound plate 2 Buffer reservoir 16 System Description IonWorks 2 0 software requires the components to be ordered as in this list and as shown in below with the Solvent Boat in the position that the Buffer Station was previously
38. 3 during most of assay 4 3 during Hole Test Note Typical source of leaks are vacuum waste bottles and plenum clamp a Antibiotic is used in the antibiotic bottle during assay runs For flushing and rinsing internal solution is used in the antibiotic bottle 132 Maintenance 6 1 2 Prior to Each Run Tasks to perform prior to each run Step Action 1 Verify quantity of fluid in each source bottle 2 Verify that the correct reservoirs are located in the correct position 3 Verify that the compound plate is seated properly 4 Remove any covers from the compound plate s 5 Verify that all the removable reservoirs are seated properly 6 1 3 End of the Day Tasks to perform at the end of the day Step Action Empty all bottles of fluid Fill 1 2 in all bottles with 50 ethanol solution Fill buffer reservoir with 5 mL of 50 ethanol solution AJOJN Run the Flush and Rinse command Dry out area under PatchPlate Caution Do NOT touch ground electrode 6 Place a used PatchPlate in plenum 6 1 4 PatchPlate Contamination To minimize PatchPlate contamination Load PatchPlate just prior to experiment Clean cell and buffer reservoirs prior to experiment o Wash with deionized water 133 User s Guide for lonWorks Quattro 134 o Blow dry with clean dry compressed air 6 2 Rechloriding the E Head Probes As part of routine maintenance the 48 silver
39. Dispense Check to aspirate each compound delivery to the PatchPlate independently rather than aspirating sufficient compound in a single draw for four wells This improves the dispensing precision for the four replicate wells This option is only enabled for the 12 channel pipettor when dispensing quadruplicates Note f Singleton Dispense is used with Use Extra Aspirate the extra aspirate volume is drawn with each transfer not divided among all four singleton transfers Singleton Dispense adds roughly 2 5 minutes to the total runnina time of a PatchPlate shown in System Software Item Description the Measurement Timing vs Scan Type table Enable for use with sticky compounds that may adhere to the interior surface of the pipettor tips The pipettor aspirates a volume of compound equal to the volume to be transferred to the PatchPlate and then holds that volume for the time entered in the Duration field Expel to After holding the compound the pipettor can either Waste Expel the volume to waste in the pipettor wash station then return to the compound plate Pipettor Prewetting Compound Plate Mix the aspirated compound Option back into the compound plate Set the number of mix cycles in the Mix Cycles field In either case the pipettor then aspirates the transfer volume and moves to the PatchPlate to complete the compound transfer Note The location to perform prewetting is ena
40. However for more reliable and consistent vacuum performance it is preferable to use the vacuum pump supplied with the lonWorks Quattro system Introduction 1 3 Safety 1 3 1 Documentation User Attention Words Four user attention words appear in the text of all Molecular Devices Corporation user documentation Each word implies a particular level of observation or action as described below Note Calls attention to useful information Important Indicates information that is necessary for proper instrument operation Caution Cautions the user that a potentially hazardous situation could occur causing injury to the user or damage to the instrument if this information is ignored WARNING Warns the user that serious physical injury to the user or other persons could result if these precautions are not taken Chemical Hazard Warning WARNING CHEMICAL HAZARD Some of the chemicals used with Molecular Devices instruments are potentially hazardous and can cause injury or illness Read and understand the material safety data sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials Minimize contact with and inhalation of chemicals Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or clothing For additional safety guidelines consult the MSDS Do not leave chemical containers open
41. Place position in the lonWorks Quattro into the Plate 2 position in the system lonWorks Quattro system Prime a clear bottom 384 well test plate with 40 uL of DI Water Pre read the test plate in a standard absorbance reader at 426 nm Place the test plate into the Plate 1 position in the lonWorks Quattro system Select Test Pipettor from the Utilities menu and set the desired test parameters in the Pipettor Validation dialog box Ensure correct Dye amp Read Plates are selected from the drop down menus After the test is completed the test plate should be covered shaken for approx 12 min and left to rest for 12 min to allow for complete mixing of the dye Read the test plate in a standard absorbance plate reader at 426 nm Detailed instructions 0110 0630 SoftMax Pro templates and Excel analysis templates are available on the lonWorks Quattro v2 0 software CD SoftMax Pro templates SoftMax Pro templates 8700 0001 LoCap 8700 0068 LoCap 8700 0002 HiCap 8700 0069 HiCap Excel Analysis Template Excel Analysis Template 8700 0027 8700 0058 Maintenance 6 5 Washing the Pipettor The Wash Pipettor utility allows you to combine several types of washing in a single process The utility provides options for washing at the solvent boat buffer boat and the wash station locations How to use the wash pipettor utility Step Action Place appropriate cleaning solutions in the solvent boa
42. Prepare and Load the Perforation Agent Prepare the perforation agent as described in Preparing the Perforation Agent on page 111 and replace the contents of the Antibiotics source bottle with the perforation solution 118 Operating the System 5 10 2 Check Solutions and Stations Check to be sure that All source bottles are filled with the correct solution types All source bottles are filled with sufficient solution volumes 5 10 3 Preparing and Loading the Compound Plates To prepare and load the compound plates Step Action Prepare the compound plate Caution Check and verify that the system software is configured for the correct plate 96 or 384 well type and the corresponding compound plate station plate 1 or plate 2 The 12 channel pipettor is compatible with 96 and 384 well plates whereas the 48 channel pipettor is compatible only with 384 well plates Failure to configure the software or to specify the correct plate type will damage the pipettor Load the prepared compound plate into the correct compound plate position Caution Check and verify that the compound plate is loaded in the correct position fully seated in the experiment deck and all plate lids have been removed 5 10 4 Preparing and Loading the Cells The following reagents and supplies are required to prepare the cells ltem Source Versene GIBCO P N 15040 066 i GIBCO DPBS P N 14040 or external buffer of Media c
43. Reservoir The cell suspension station holds cell suspension for normal experiments in a single well V bottom reservoir or a multi well cell boat can be used for cell line optimization During an experiment the 12 or 48 channel pipettor aspirates the cell suspension from the station and dispenses it into the PatchPlate This station is labeled CELLS on the experiment deck 2 3 8 Solvent Boat A four slot solvent boat is provided for dual addition experiments Single addition experiments can use either the solvent boat or the second compound plate position for an optional solvent wash step The solvent boat contains four slots with 4 mL capacity each The pipettor washes in one trough of the solvent boat for all of the solvent wash cycles for a given compound transfer That is on systems equipped with a 12 channel pipettor the first slot will be used for compound transfers from rows A and B from a 96 well compound plate the second slot for rows C and D etc On systems equipped with a 48 channel pipettor the pipettor washes in the first and third slots for rows A C of the 384 well compound plate and the second and fourth slots for rows B D For the remainder of the compound plate the pipettor returns to the first and third slots for rows E G and the second and fourth slots for F H The same patterns repeats for rows J P thus for single addition experiments with many solvent wash steps there is greater likelihood of compound contamin
44. S No Seal FO Filtered Out MR Manually Rejected This option replaces the code with a tab to keep the spacing of the values in the columns correct Enter a name for the metrics file to be exported Select Filename and Path and open the Browse dialog to select a directory in which to write the file 4 5 3 Export Trace Images Images of selected trace data can be exported from an open data file or from the Trace Groups View window Data File Trace Display Area Select Export Dialog from the graph popup menu in the trace display area of a data file 103 User s Guide for lonWorks Quattro Trace Groups View Select Export Dialog from the graph popup menu in the Trace Groups View window Both graphs use the same Exporting or Exporting Cells dialog Item Export Description Select the file format in which to export the image Options include Windows MetaFile BMP JPG and PNG There is also an option to export the file in text format Export Destination Select to export the image to the clipboard as a file or send it to the printer If you select to export as a file you must select a directory to write the file to Name the file in the same Save As dialog Object Size You can optionally specify the size of the image in millimeters inches or pixels 104 Operating the System Chapter 5 Operating the System This chapter provides information and procedures required to o
45. This becomes gray if settings are changed press the Apply button at the bottom of the dialog to update it The sections below cover each of the tabs in the dialog as well as additional dialogs opened from them 4 3 2 File Saving Tab Use this tab to set a naming convention for data files generated when the protocol is run and to select a folder to write the files to Item Description Date Prefix Automatically names files with the date they are generated in format MMDDYYYY_n with incrementing n number for each file generated in a day Custom Prefix Type in your own file name This automatically has _n appended numbering off each new file generated File Naming Prompt at Run Time You are prompted at the start of each experiment using the protocol to enter a name for the data file 53 User s Guide for lonWorks Quattro 54 Item Description Save Experiment Data Files to Opens a Browse dialog to select the folder for data files to be written to If the selected folder is on a network drive the Application will save the file first to a temporary folder on the local disk and then move it to the network when the experiment has completed This feature allows the instrument to complete the cleanup step quickly and provides a safe location in case the network is unavailable Note If the file cannot be moved to the network it will remain in the temporary folder and must be manually cop
46. User s Guide for IonWorks Quattro wow Molecular Devices Molecular Devices Corporation Sunnyvale California 94089 Part 0112 0125 Rev A Copyright 2005 Molecular Devices Corporation 12 05 0112 0125 Rev A This manual contains information protected by copyright No part of this manual may be photocopied or reproduced in any form without prior written consent from Molecular Devices Corporation Trademark Information IonWorks is a registered trademark of Molecular Devices Corporation MDC and is not to be used in any type of promotion or advertising without permission from MDC All other trademarks are the property of their respective owners Molecular Devices Corporation Disclaimer Molecular Devices Corporation MDC makes no warranties express or implied including without limitation the implied warranties of merchantability and fitness for a particular purpose regarding IonWorks MDC does not warrant guarantee or make any representations regarding the use or the results of the use of IonWorks in terms of its correctness accuracy reliability currentness or otherwise and the entire risk as to the results and the performance of IonWorks is assumed by you The exclusion of implied warranties is not permitted in some states therefore the above exclusion may not apply to you In no event will MDC its directors officers employees or agents be liable to you for any consequential incidental or indi
47. a chloride coating to the electrodes see Rechloriding the E Head Probes page 134 The soak station is labeled SOAK on the experiment deck 2 3 3 Electronics Head Wash Station This station is an embedded non removable reservoir that contains 48 wash channels for individually rinsing the 48 probes of the electronics head A peristaltic pump brings wash solution from the E Head Wash source bottle while a solenoid pinch valve connected to the system vacuum line opens an exit drain connected to the Wash Waste bottle 2 3 4 PatchPlate Station with Clamp Frame The PatchPlate consumable is loaded in the PatchPlate station where all experimental operations fluid pipetting plate priming fluid exchange and electrical recording occur A clamp frame vacuum seals the PatchPlate consumable to the station System Description The mechanical block that houses the PatchPlate is called the plenum The PatchPlate cavity in the plenum houses a ground bath electrode and a small port for monitoring the plenum vacuum pressure as well as for transporting plenum fluidics into and out of the ports The top surface of the plenum has an embedded o ring and a vacuum port The o ring sits around the perimeter of the PatchPlate cavity and seals the PatchPlate to the plenum A switch connected to the vacuum port controls the PatchPlate clamp frame When activated the vacuum port seals the clamp frame to the plenum securing the PatchPlate The PatchPlate
48. about 10 12 seconds for the process to complete When the dialog indicates the Firmware has terminated click the Update Firmware button A message will quickly appear indicating the Firmware has been updated Power down the unit as instructed wait about a minute and then power the unit back up 10 Close the Firmware Upgrade Utility dialog The green connect light will be dark 11 When the unit has finished rebooting the green connect light will come on again The unit is ready for use Technical Assistance Appendix A Technical Assistance If you need help to resolve a problem there are several ways to contact Molecular Devices World Wide Web www moleculardevices com Phone 800 747 5577 Fax 1 408 747 3601 E mail info moldev com Questions See the Molecular Devices Knowledge Base www moleculardevices com pages support html 139 Index 48 Channel Electronics Head 22 Active Protocol Summary 45 Cleaning the System 7 Command Voltage Compound Plates scc Conditioning Train Configure Metric Export Data EXpOrt ceceeeseenesees 100 Data Files cccccceseeseeseneee Data Menu Electronics Head Mechanism OLA CHOR i nera tA 22 Electronics Head Soak DtatlONs idee elaine 18 Electronics Head Wash Stalina eni Sotese Experiment Deck Experiment Deck Experiment Menu Experiment Protocol SUMIMALY siirre 46 Exporting Data 123 Expor
49. age Select to order by row or column There will be one row or column per scan per metric Select Metrics to Include Select individual metrics to export by highlighting them in the list Click a second time to deselect a metric All User Defined Only Select just the user defined metrics This excludes the Resistance and Offset Intercept mV voltage metrics Exclude Hole amp Seal Test Results of the hole and seal tests are not included in the export file Select Layout and Style Order the data in the exported file and select System Software Section Description what additional information to show Protocol Info Include no protocol information or add a terse or verbose description of the protocol at the end of the export file Metric Order Select the ordering of the metric values within the general layout defined in the format section For example with Compound Plate format Metric Scan Cell groups results first by the metric then scan and finally by cell As an example with four metrics and three scans there would be four sections one for each metric and within each section the four cell replicate columns would be grouped together by scan Suppress column headings Hide column headers Suppress non numeric results Wells that fail either the Native or User Defined filters or are manually rejected do not export a value Instead one of the following codes is used HB Hole Blocked N
50. aps will result in linked tubing lines Check that all bottles are seated properly in the holding rack positions All bottles contain a flange at the lower part of the bottleneck This flange supports the weight of the bottle as it is inserted into the holding rack Improper seating will damage the bottles and connected tubing 11 Load a used PatchPlate into the PatchPlate station on the experiment deck Check and if necessary dry any traces of fluid on the top surface of the PatchPlate plenum Caution Keep the top surface of the plenum dry at all times The top surface of the plenum houses the vacuum port and introducing moisture will damage vacuum pump Place the PatchPlate into the plenum cavity Seat the clamp frame on the PatchPlate station Turn the PatchPlate vacuum switch from the Release to the Hold position Apply an even force to the clamp frame to create a vacuum seal Note Ensure that there is no audible air leak e g hissing at the clamp interface 12 Close the instrument access door and turn the handle to the lock position 13 Wait for the traffic light icon on the main window toolbar to turn green 14 From the Utilities menu select flush and rinse The system will perform a flush and rinse cycle to clean the fluidics lines and the 12 channel pipettor 15 16 17 After completion of the flush and rinse cycles open the instrument access door and remove the v b
51. at a different row of the solvent plate after each row of the compound plate After washing in solvent the pipettor is washed as usual in the pipettor wash station The solvent wash plate should have sufficient volume in the wells to exceed the dispensed compound volume 77 User s Guide for lonWorks Quattro 78 Item Description Wash Cycles Specify the number of solvent wash cycles up to 20 Each cycle consists of one aspirate and one dispense of solvent in and out of the pipettor Location Select the location of the solvent You can use a plate location if solvent is in a standard size plate or the solvent boat Note The plate location is automatically set to be the location other than the location selected for compound If a boat type plate is used it must be added to the plates txt file and properly calibrated The pipettor will move to different positions within a boat type plate Pipettor Transfer Options Wash Cycles Select the number of standard wash cycles up to 20 used to wash the pipettor after each compound transfer sequence Compound Mix Cycles Select the number of compound mix cycles from 0 to 10 to mix compound before aspirating compound for delivery to the PatchPlate Use Extra Aspirate Check and enter an additional volume of compound to aspirate to give highest pipetting performance Setting extra aspirate marginally increases experiment time Use Singleton
52. ation in the solvent boat and it is System Description therefore preferable to use the second compound plate position instead The second compound plate is unavailable in dual addition protocols so the solvent boat is the only available option under this circumstance 2 3 9 Buffer Reservoir The BUFFER station houses a removable V bottom reservoir used to hold buffer solution to be dispensed into the PatchPlate during an experiment The buffer station is also used for rinsing the pipettor during flush and rinse operations 2 4 12 or 48 Channel Pipettor The pipettor which can be fitted with either 12 or 48 needle heads dispenses buffer cell suspension and compounds into the PatchPlate The 12 channel pipettor can be used with 96 and 384 well compound plates whereas the 48 channel pipettor is compatible only with 384 well compound plates The pipettor can be used to load either all or a portion one half one quarter or one eighth of the 384 wells in a PatchPlate consumable Depending on the chosen PatchPlate coverage operations such as loading cells may require pipetting into all 384 wells The pipettor has the capacity to load all 384 wells of the PatchPlate consumable in a single aspirate and multiple dispense cycle The pipettor can also perform mixing functions 2 4 1 Pipettor Mechanism of Action The design of the pipettor incorporates 12 or 48 pistons that move in individually isolated cylinders Each cylind
53. bled only for Dual Addition experiments The mix cycles option can be used to remix compound before transferring by setting Duration to zero Enter voltage offset correction values to be subtracted from pre compound and or post compound scans Liquid Junction Potential Correction 4 3 9 Analyze Export Data Tab Use the Analyze Export Data tab to configure metrics measurements taken from specified regions of the command voltage response in your protocol An option allows you to take two different measurements from each scan and combine these in a simple relation such as ratio or difference For family command voltages you can make comparisons of measurements 79 User s Guide for lonWorks Quattro 80 taken in the different member scans of the family See User Metric Editor section following for details Metrics configured in other protocols or data files can be imported into the current protocol or data file You can also set up automatic export of the metrics you configure and optionally export the data traces recorded The metrics of already recorded data files can be viewed and edited from the User Metric List Editor opened with the Edit User Metric List command This dialog offers the same options as those on the Analyze Export Data tab in the Protocol Editor dialog Item Description User Defined Metrics Use the buttons to manage the metrics included in the protocol or data fil
54. bottle Installing the incorrect bottle cap to the source bottle will result in flow of incorrect solution to various stations O Important Check that all bottles are seated properly in the holding rack positions All bottles contain a flange at the lower part of the bottleneck This flange supports the weight of the bottle as it is inserted in the holding rack Improper seating of the bottles will damage the bottles and the connected tubing From the Utilities menu select Flush and Rinse Repeat this command a second time if there is residual ethanol from previous cleaning 5 8 Preparing the Experiment Deck The experiment deck is the area on the instrument surface that contains the PatchPlate plenum cell and buffer reservoirs compound plates E head pipettor and solvent wash stations and E head soak position for rechloriding electrode pins see page 134 5 8 1 Loading a PatchPlate To load the PatchPlate Step Action 1 Open the instrument access door 2 Check that all stations with removable reservoirs and compound plates are empty Load a used PatchPlate into the PatchPlate station a Check and if necessary dry any traces of fluid on the top surface of the PatchPlate plenum Remove the used PatchPlate Caution Keep the top surface of the plenum dry at all times The top surface of the plenum houses the vacuum port Avoid introducing any moisture into the vacuum port as moisture w
55. cans with Single Addition To configure repeated scans with a single compound addition select Repetitive Scans with Single Addition in the experiment type options in the Add Compound Measure Currents section and then press Scan Parameters Editor This opens the Set Repetitive Scan with Addition Parameters dialog This experiment type is a combination of the fixed scanning experiment and the standard single addition experiment The dialog allows the user to select plate type incubation time and addition volume similar to the Single Addition experiment and then a number of scans to be acquired both before and after the addition This experiment type only allows the Eighth at Once EAO scan mode so that the E head remains in the wells between the scans to keep the cells at the holding potential Delay Between Scans applies only between successive scans not during the compound addition phase of the experiment 75 User s Guide for lonWorks Quattro 76 Based on the plate coverage and the number of scans to be acquired the dialog estimates the total measurement time and the final size of the experiment file The user should note that this experiment type can create very large experiment files if a whole PatchPlate is used with a large number say 20 or more of scans The maximum number of scans either before or after the addition is fifty Such an experiment can result in a file size greater than 1 GB and is likely to cause low virt
56. ch on the experiment deck is set to Release All source and waste bottles have been removed and cleaned A used PatchPlate is positioned in the plenum to exclude dust or particulates The instrument access door is closed and the handle is turned to the lock position 35 System Software Chapter 4 System Software This chapter provides a detailed description of the features and functions of the lonWorks Quattro System software Topic See Page Interface Overview 38 Menu Bar 42 Protocols 52 Data Files 86 Data Export 100 37 User s Guide for lonWorks Quattro 38 4 1 Interface Overview The main window of the IonWorks software has a title bar menu bar and toolbar at the top and a status bar along the bottom Data files open into the large central work area The basic interface layout is illustrated in Figure 6 Title Bar Menu Bar Toolbar Main Work Area Data File Window Status Bar Figure 6 Overview of the software user interface In addition dialogs opened from the menus or toolbar are used to configure experiment settings Most important of these is the Protocol Editor dialog for configuring the current protocol that will control an experiment when the IonWorks Quattro is run This is opened from Edit Protocol or the Edit Active Protocol toolbutton Short descriptions of the main features of the u
57. d Description Flush and Rinse Flushes the tubing in the vacuum system and rinses using buffer See Daily Maintenance page 132 for instructions on use Vacuum Pressure Check Runs a test of the plenum vacuum pressure and displays the result in a dialog box Wash Electrodes Moves the E head to the wash station and executes the wash process Rechloride Electrodes Opens the Rechloride Electrodes dialog See Rechloriding the E Head Probes page 134 for instructions on use Park Electrodes Moves the E head back to the home position Test Electrodes Opens the Test Electrodes dialog See Detecting a Salt Bridge page 135 for instructions on use Wash Pipettor Opens the Wash Pipettor dialog See Washing the Pipettor page 137 for instructions on use Park Pipettor Moves the pipettor back to the home position Test Pipettor Opens the Pipettor Validation dialog See Testing the Pipettor page 136 for instructions on use Stop Stops the utility operation in progress 49 User s Guide for lonWorks Quattro 50 Command Description Firmware Version Check Displays the version of the currently running Instrument Firmware in a dialog box Shut Down Firmware Forces a shutdown of the Instrument Firmware The instrument must be powered down and back up to restart the firmware Upgrade Firmware 4 2 6 Data Menu Opens the Firmware Upgrade dialog S
58. d the active protocol can be loaded ready to run at a time The name of the current active protocol is displayed in the Status bar Protocols must be loaded in order to configure them or to run an experiment Load saved protocols from File Open Protocol or configure new protocols with File New Protocol New protocols are called default until saved and named Saving options are also in the File menu Protocols are configured in the Protocol Editor dialog opened from Edit Active Protocol or the toolbutton A concise summary of the active protocol can be opened from the View Active Protocol Summary command or use the toolbutton When viewing the summary you can open the Protocol Editor dialog directly from the summary window Edit Protocol in the summary window menu if you want to edit it 4 3 1 Protocol Editor Dialog The Protocol Editor dialog has four tabs on the left used to configure the protocol 52 System Software File Saving Set file naming and saving options for data files generated by the protocol Start Seal Test Basic settings to prepare cells for the test procedure Access End Settings for the patch clamp experiment including compound additions and command waveform configuration Analyze Export Data Configuration of analysis metrics for the resulting data and automatic export options The right hand section of the dialog has a summary of the current settings
59. data is stored in an ASCII text file format for input into a spreadsheet or database program 1 2 IonWorks Quattro System Specifications and Requirements 1 2 1 Electrical Requirements Item Parameter Description Value Line Voltage 120 VAC or 230 VAC instrument versions Note System power supply is configured when manufactured User s Guide for lonWorks Quattro 1 2 2 Physical Specifications and Requirements Item Parameter Description Value Instrument dimensions The lonWorks system cabinet is 1 412 m 55 6 in high x 1 476 m 58 1 in wide x 0 826 m 32 5 in deep with access door closed Space requirements An additional 0 457 m 18 in is required on each side to accommodate the source and waste container holding racks There should be a minimum 0 152 m 6 in clear at the back of the instrument 0 813 m 32 in is required to allow clearance for opening the access door Additional setup space An additional benchtop 0 914 m 36 in wide 0 762 m 30 in long and 0 914 m 36 in high is required for the host computer display monitor and peripheral accessories 1 2 3 Vacuum Requirements The IonWorks Quattro requires vacuum to operate This can be provided by house vacuum or by purchasing a vacuum pump House vacuum must meet the following specifications 120 mb 0 6 m hr or 26 in Hg 0 35 cm Have fittings required to attach to a 1 4 plastic tubing
60. e Metrics are listed in the field in the left of the section New Metric Opens the User Metric Editor dialog see below to configure a new metric Edit Metric Opens the User Metric Editor dialog see below populated with the settings of the currently selected metric allowing you to edit the metric Delete Metric Delete the currently selected metric Import Metrics Opens the Metric Import dialog to browse to lonWorks protocol inp or data ihe files Click on a protocol or data file to show the metrics defined for it in the right hand field Press the Import button to import the metrics you must import all the metrics defined for the selected protocol or file If the metrics in the selected file are not compatible with the data sets in the current data file a warning will appear in the box instead Automatic Metric Export Check to automatically export metrics data when the protocol is run Configure export options in the Configure Metric Export dialog page 101 System Software Item Description Check to automatically export trace data when the Automatic Trace protocol is run Export Configure export options in the Configure Trace Export dialog page 100 4 3 10 User Metric Editor Use the User Metric Editor dialog to configure metrics in protocols or existing data files To open the dialog Protocols Press New Metric or Edit Metric in the Analyze Export Data tab of the Protoco
61. e data files can be opened at once each in its own window Standard Windows buttons in the top right hand corner of data file windows Maximize files to occupy the entire central work area Minimize files to an icon in the bottom left of the central area Allow manual resizing of the window Data files cannot be moved outside the central work area of the main interface If multiple files are open at once they can be cascaded or tiled using options in the Window menu The Window menu also contains a list of all open files click on a file in the list to bring that file to the top The main work area of the lonWorks interface is gray at default Windows color settings when the program is first opened before any data files are opened 4 1 5 Status Bar The Status Bar reports The currently loaded protocol file use File menu options to change the protocol file if you want The currently selected pipettor 12 or 48 channel set from Experiment Set Pipettor Type The type of PatchPlate loaded i e o Standard or 41 User s Guide for lonWorks Quattro o PPC Population Patch Clamp The Status Bar reports the PatchPlate type that is currently selected in the active protocol Important The PatchPlate type must be manually selected in the Protocol Editor by the user prior to commencing the experiment The Status Bar can be toggled in and out of view from the View menu 4 2 Menu Bar The
62. e lonWorks Quattro system Contact Molecular Devices to request additional plate definitions 384 Well Plate Settings When a 384 well plate is selected as the compound plate select the starting well A1 A2 B1 or B2 to define the quadrant from which to commence pipetting See supplemental Compound Plate Maps PDFs located in the Help menu Compound Locations and Dispense Volumes Compound Location Select plate 1 or 2 as the compound plate If you have the other plate location selected for solvent its location automatically changes if you change this setting The selected plate locations are reported at the top of the dialog beside the graphic Volume of Compound Select the volume of compound to be dispensed 3 0 3 5 4 0 5 0 or 6 0 microliters Volumes of buffer solution and cell solution are automatically adjusted depending on this setting to give 2x or 3x dilution as shown in the table the default setting is bolded Compound Buffer Cells Dilution ae 3 0 3 0 3 0 3x 9 0 3 5 3 5 3 5 3x 10 5 4 0 2 0 2 0 2x 8 0 5 0 2 5 2 5 2x 10 0 6 0 3 0 3 0 2x 12 0 Buffer and cell volumes are reported at the bottom of the section in the dialog 67 User s Guide for lonWorks Quattro 68 Item Description Select Timing and Scan Parameters Compound Incubation Time Enter the length of time that you want the cells incubated with compound before applying the vol
63. ect more than one data set hold the CTRL key down while selecting the desired data set in the list To customize the filename prefix edit the text in the Export File Prefix 5 field For PatchPlate wells the software will append _RCC_PP ihd where R is the row letter and CC is the column number For Compound Plate wells the software will append _RCC_CP ihd 6 To change the file destination click Browse to open the Browse for Folder dialog box Navigate to the desired directory and click OK When finished click OK in the Configure Trace Export dialog box A 7 message will appear while the data is being processed to the tab delimited format 5 12 Importing a Data File into Microsoft Excel Data from single or multiple wells may be imported into a spreadsheet application such as Microsoft Excel if it has been exported in a tab delimited format Opening the data file within Microsoft Excel will launch the File Import Wizard which will guide you through the process 125 User s Guide for lonWorks Quattro 126 5 12 1 Importing Data Files To import data files into Microsoft Excel Step Action 1 Open Microsoft Excel and select Open from the File menu In the Open File dialog box navigate to the data file location 2 Note Make sure that All Files in the Files of Type field is selected Select the file or files that you wish to open 3 Each file comprises the data from
64. ee Upgrading the Instrument Firmware page 137 for instructions on use Set filter and correction options for raw data traces in data files from this menu Note This menu is only available when a data file is open Item Description Leak Correction Applies or removes leak correction to all pre and post compound scans in the current data file Leak is estimated from the response to the pre signal step automatically collected at the beginning of each waveform scan and a correction is applied to all values in the scan adjusted according to the command voltage delivered at each time point Offset Current Opens the Configure Offset Current dialog to correct for minor differences in the non stimulated ion current of the cells Select an interval of the command voltage where the cell is not stimulated e where you expect zero current response The mean value of the response from this range in each scan is then set to zero Note The Apply Offset Subtraction correction is a display correction only it does not alter the original data This feature is disabled by default System Software Item Description Boxcar Filtering This toggle command turns boxcar filtering on and off The width of the averaging window is five data points This setting is not user adjustable This feature is disabled by default 4 2 7 Window Menu A description of the Window menu Commands is listed
65. ee i oe n Ea saei S 109 5 4 Preparing Buffer SOlMtoms s ccccscasscecsecisdetstsscsssiseieentetecvesssiesdobotscesvevaseseatasteonensadeocodganensu 109 BAN External Buffer oniinn r A E E A E E Ee 109 54 2 Internal BUMMER miripis irai AE E EN RERE EEE 110 5 4 3 Determining Liquid Junction Potential Correction voltage values cceeee 110 5 5 Preparing the Perforation Agent 0 ccccccscsesssteeeseeeeteteesesesnsnensnesssssceceseeeneeneneseseananens 111 51 Using Amphotericin B oisein ieren e a ppe a a deans E ATRE Eara 111 5 6 Preparing Compound Plates asi si ses asinine canst dian nlaneak sti A 112 5 6 1 When to Prepare the Compound Plate ccccccccsssecsseeeetetetesesestenenenesesseeeeeseeeenenes 112 5 6 2 Compound Plate TyPes cccccesessesssesesesssnsneensssecececeeeesnenesesesessenenenesssseceeeseeeenenes 112 5 6 3 Dispensing from Compound Plates 0 0 ccc ceeeeeseneteesesesnenenenesssseceseseeeenenes 112 5 6 4 Concentration of Compounds in the Plate ccccccesesesssneseseseeeeeeeeneteteeseseanenenes 113 5 7 Preparing the Fluidics System smrce niers e ree ieri ia e iieii 114 5 8 Preparing the Experiment Deck ccececcccseseesetesesesesnenenesesesesnenenesessecececeeenenenenesssesnanens 116 5 8 1 Loading a PatchPlate sinerien tei ienien EEEE Ei Ee rE E E e 116 5 8 2 Preparing the Fluid Lines and 12 or 48 Channel Pipettor cccceeeeeeeeeeees 117 5 9 Setting Up the Experiment Protocoleseresisen oraiiio nn ie a 118 5 9 1
66. em is connected to house vacuum ensure that it is on and operating properly Turn on the system computer and monitor Log in to the system and launch the lonWorks Quattro software by double clicking the desktop icon Turn on the instrument with the power button on the front of the power supply module in the electronics cabinet Starting Up and Shutting Down the System Step Action 10 Wait until the ready traffic light button is lit green in the software main toolbar before proceeding This will take 3 5 minutes 11 If performing an experiment proceed to Chapter 5 Operating the System page 106 3 2 Shutting Down the IonWorks Quattro System Note This procedure should be followed after an experimental run and the system has been properly cleaned as described in Chapter 5 Operating the System To shut down the IonWorks Quattro system Step Action At the end of the programmed experiment wait for all operations to terminate The ready traffic light on the main window toolbar should be green Check to make sure that a Flush and Rinse operation was performed with 50 50 ethanol filtered water wash solution see Chapter 5 Operating the System page 106 Exit the lonWorks Quattro software by selecting Exit from the File menu If not using house supplied vacuum turn off the vacuum pump Check and verify the following The PatchPlate vacuum swit
67. emoving and collecting air bubbles from the plenum tubing lines Referred to as debubblers these vessels are connected to the computer controlled vacuum regulator that is integrated with the fluidics system Consequently the debubblers serve as control points for regulating the plenum differential pressure The upper debubbler is located at the left side of the experiment deck and can be used as a quick user feedback for monitoring the plenum fluidics operations After completion of priming debubbling and or fluid exchange operations the fluid height at the upper debubbler must reach the appropriate level Fluid heights that fall outside the appropriate range may indicate a problem with the fluidics system 2 8 3 Vacuum Regulator The integrated vacuum regulator adjusts the vacuum pressure to required levels for priming the PatchPlate and adding cells A digital vacuum gauge on the fluidics system displays the actual differential vacuum pressure inches of water The vacuum is also used to empty the contents of both the pipettor and the electronics head wash stations 2 9 Source and Waste Bottle System The source and waste bottles are located at the left and right sides of the system cabinet respectively The source bottles supply the system with fresh solutions while the waste bottles collect the fluid waste 28 System Description 2 9 1 Source Bottles Four source bottles are housed on the left side of the system cabinet The table
68. er is sealed at one end and open at the other The underlying mechanism for the operation of the pipettor is air displacement As pistons move within the cylindrical housing air is displaced The air displacement either creates a vacuum for aspirating fluid 21 User s Guide for lonWorks Quattro 22 into the individual tips or exerts a force that dispenses the fluid out the individual tips The motion of the pistons is controlled by stepper motors and encoders to maintain the required pipetting performance Note The pipettor performance is designed to meet the precision and accuracy required of industry standard micro pipetting systems 2 5 48 Channel Electronics Head The 48 channel electronics head records electrical measurements from the 384 wells of the PatchPlate The head is comprised of 48 silver plated probes that access individual wells of the PatchPlate to measure electrical currents simultaneously Each probe is connected to a separate amplifier on an enclosed printed circuit board inside the electronics head housing Each electronics head is calibrated across all 48 probes to normalize performance A silver chloride coating is applied to the probes periodically by soaking them at the soak station The electronics head is equipped with motion axes that allow the head to access either the PatchPlate or the electronics wash and soak stations Caution DO NOT CONTACT any part of the electronics head The head housing
69. es to be made to the currently active protocol see page 52 Make Experiment Protocol the Active Protocol Save Experiment Protocol to a File Makes the protocol from a saved file the active protocol Opening a new file does not automatically update the active protocol Saves the protocol portion of an open lonWorks data file to disk without making it the active protocol View Active Protocol Summary Opens the Active Protocol Summary window see page 45 which reports configuration details of the currently loaded protocol reported in the Status Bar View Experiment Protocol Summary Opens the Experiment Summary window see page 46 with protocol configuration details of the current data file View Traces Opens the Trace Groups View window see page 46 to display signal traces from the current data file Edit User Metrics List Opens the User Metric List Editor see page 81 Edit Native Filters Opens the Native Filter Editor dialog see Edit menu page 44 Messages and Logging Opens the lonWorks Messages dialog which displays status messages from the instrument during operation Messages can be optionally logged See page 48 Start experiment Starts the experimental protocol Stop experiment Stops the experimental protocol System Software 4 1 4 Main Work Area The main work area of the lonWorks interface is used to display data files Multipl
70. esesneneenesssseees 6 1 2 3 Vacu m Requirements sieis srnri eissis iniiae eirean nae i i S E ESE ER E e ia 6 NI Saf EY spaced N E EEA EEEE T AE ea A eaa tenes 7 1 3 1 Documentation User Attention WordS esesesssrssseerstesrsestestesstesserstsnteseestenteseeestentes 7 Chemical Hazard Warning ission uani ei a geire 7 1 3 2 Chemical Waste Hazard Warning cccccsesssssseescseeeeseeesenesesesesesnenensnesessscececenenssesees 8 1 3 3 ABOU MSD Soo eE EEEE KEE EEEE E E AEEA ER 8 13 4 Ordering MSDSs griy EEEE EEE EROE EEE E EET aE 9 1 3 5 Instrument Safety Labels soreness s iien Ne e aE EEEE NE i s 9 1 3 6 Before Operating the Instrument es sessssseseerttsrsesreertsrtesesestesntesseestsnteseestentesetestentes 9 14 Saftety Alert Symbols asotis eao e EEEE EE E EE ER 10 TAA Electrical Symbols eiisnie e a a ae aR 10 14 2 Nonelectrical Symbols e R E E a E oa sees 11 Chapter 2 System Descriptio ssie eesse isiessesii sosatt Sesnessi saos ross nensis ie aeoi senose Sees s iest So si irets 13 2 1 IonWorks Quattro System COMPONENMs cceccceesesesneneesesesesneneesssseeeceseeeneneesesesesnenens 14 2 2 System Cabinets corister css En EE eA aae Ta E bevesnerstio sd EREE sheesh 15 iii User s Guide for lonWorks Quattro 23 Experiment DOCK ieii a E ER tated stgtte Riera eee eae Guat 16 2 3 1 Components of the Experiment Deck cccccccsssseseteseseseeneneseseseeeeeeceeeenesesesesnenenenes 16 2 3 2 Electronics Head Soak Station
71. g With dual compound experiments three scans or three sets of scans are run before between and after the compound additions A graphic at the top of the dialog helps identify plate locations in the IonWorks Quattro In the fields below select plates for compound and solvent and configure compound additions mixing and washes You can prewet pipettor tips to help avoid losing sticky compound and set voltage offset correction Importantly set how much of the PatchPlate to deliver compound to before scanning you can apply compound to the entire PatchPlate and then proceed to scan it or apply compound to sections and scan after each Smaller sections mean cells spend less time on average in compound before scanning however at the cost of longer overall experiment time Item Description Select the plate types for the two compound plates Plates must have the same number of wells Select Plate Types Set the order of the plates that are used in the Compound Location field in the next section 71 User s Guide for lonWorks Quattro 72 Item Description The Plate Type list has predefined compound plates used with the lonWorks Quattro system Contact Molecular Devices to request additional plate definitions 384 Well Plate Settings When a 384 well plate is selected as the compound plate select the starting well A1 A2 B1 or B2 to define the quadrant from which to commence pipetting See s
72. gly recommend that you replace the MSDSs in your files each time you receive one packaged with hazardous chemicals Introduction CHEMICAL HAZARD Be sure to familiarize yourself with the MSDSs before using reagents or solvents 1 3 4 Ordering MSDSs To order an MSDS for a chemical call the chemical manufacturer 1 3 5 Instrument Safety Labels Safety labels are located on the instrument Each Safety label consists of a Signal Word panel which implies a particular level of observation or action e g CAUTION or WARNING If a safety label encompasses multiple hazards the Signal Word corresponding to the greatest hazard is used Message panel which explains the hazard and any user action required Safety Alert symbol which indicates a potential personal safety hazard 1 3 6 Before Operating the Instrument Ensure that everyone involved with the operation of the instrument has Received instruction in general safety practices for laboratories Received instruction in specific safety practices for the instrument Read and understood all related MSDSs Caution Avoid using this instrument in a manner not specified by Molecular Devices Corporation While the instrument has been designed to protect the user this protection may be impaired if the instrument is used improperly User s Guide for lonWorks Quattro Use of controls or adjustments or performance of procedures other than those specified herei
73. greater than three decimal places can be specified for the hits threshold Set Filters Once filters are defined you can apply them all at once selectively individually or in any combination or none at all using the buttons in the Filters section title bar The buttons remain in view when the section has been collapsed Press an individual numbered button to turn on the filter of that number Apply two or more filters by pressing the buttons of each of the filters The left most button turns off all filters altogether Summary Configure analyses on your data using metrics in the Summary section The results of the current Summary configuration are shown in the Plate display in the tooltips that open over each well and in the Statistics box 95 User s Guide for lonWorks Quattro 96 there The statistics values reported in the Plate display Statistics box are also written to the Statistics section below Note that summary definitions use metrics applied to the data traces after any data modification currently enabled Trace data can be modified with Leak Subtraction Offset Current and Boxcar Filter all in the Data menu see Data Menu page 50 One analysis can be selected in the Summary section at once It defines a single value for each PatchPlate well in terms of Scan or scans that are measured Metric used to measure the selected scans Means of combining the metric values from each of the component
74. hPlate The E head performs another recording to check the status of the cell s seal formation i e the Seal Test The perforation agent is introduced into the plenum under the PatchPlate The perforation agent is cycled through the plenum to optimize access to the inside of the cells The 48 channel E head applies the user defined command voltage protocol to individual wells and records the ion channel activity Pre Compound Scan s The pipettor adds compounds to the PatchPlate from the first compound plate position At this point there is a waiting period for compound incubation 10 11 12 Operating the System The 48 channel E head performs electrical recordings of the PatchPlate to gather data after the compound addition i e Post Compound Scan s For dual compound addition experiments the pipettor adds compounds to the PatchPlate from the second compound plate position At this point there is a waiting period for compound incubation For dual compound addition experiments the 48 channel E head performs electrical recordings of the PatchPlate to gather data after the compound addition The IonWorks Quattro system performs a cleanup sequence to drain all the fluid lines and to wash both the pipettor and the E head 5 2 Setting Up the IonWorks Quattro System for an Experimental Run Setting up the system requires performing the following tasks Preparing cells Preparing buffer and perforation age
75. he high measurement fidelity obtained in a standard patch clamp device with the rapid measurement throughput required for high throughput screening and drug discovery The lonWorks Quattro system uses the PatchPlate consumable to provide a custom 384 well platform for obtaining simultaneous parallel electrical recordings Integration of a 48 channel electronics head and a 12 channel pipettor equips the lonWorks Quattro system with electrical measurement and fluidic handling capabilities necessary for a full fledged screening instrument The technology of the IonWorks Quattro system enables scientists to design cell based assays for monitoring the behavior of different types of ion channels 1 1 1 How the IonWorks Quattro System Works The basic objective of the lonWorks Quattro system is to perform parallel whole cell electrophysiological measurements To achieve this individual cells are positioned on small pores at the interface of two isolated fluid chambers each containing a separate electrode Both fluid chambers contain balanced salt solutions that provide the medium for flow of electrical currents The small pores serve as the only contact point and Introduction consequently the only path for the flow of electrical currents between these two isolated fluid chambers As the cells cover and seal the small pores they interrupt the direct path of electrical flow Next a perforating agent is introduced to create an electr
76. he ion channel of interest they must maintain the required physiological pH and osmotic balance for the host cell at all times For certain ion channels such as hERG better pharmacological results are obtained when the internal buffer contains 140 mM chloride 5 4 3 Determining Liquid Junction Potential Correction voltage Important Improper voltage offsets will result in absolute voltage errors and suboptimal performance Various combinations of external and internal solutions cause liquid junction potentials in the lonWorks Quattro system Liquid junction potentials defined as potential differences between two solutions occur at all boundaries between solutions of different compositions The lonWorks Quattro system zeros all the voltage offsets that arise from electrode to solution junction potentials and liquid junction potentials during the first portion of the experiment prior to the cell addition i e during the Hole Test However the Liquid Junction Potential is then at least partially removed by cell addition and occlusion of the hole s resulting in the Operating the System command voltages being off by approximately the value of the Liquid Junction Potential The final correction factor that is applied to compensate for these voltage offsets during cell recordings requires a user input value for the Liquid Junction Potential Correction The voltage offset values if significant must be determined empirically for each co
77. he read bank interleaving option Item Description Holding Potential Set the voltage the cell will be held at before and after the command voltage is applied This parameter is used to allow the cell to enter a resting state where all of the ion channels are closed Value range 1000 to 1000 mV Holding Potential Pre Sequence Time The period the cell is held at the holding potential before the scan sequence begins Post Sequence Time The period that the cell is held at the holding potential after the scan sequence has completed Kumber or Repeat the number of times that the entire conditionin Conditioning Trains pe g train is run per Scan System Software Item Description Check to have the pre signal and conditioning train delivered to all 48 channels of the electronics head simultaneously after which the command voltage is delivered to each 12 head read bank sequentially Enable Read Bank Unchecked the entire sequence of pre signal Interleaving conditioning train and command voltage is delivered to each read bank sequentially Use the View menu All Read Banks option to see the effects of this setting 4 3 6 Scan Parameters No Fluid Additions Repetitive Scans To configure scans with no compound additions select No Fluid Additions Repetitive Scans in the experiment type options in the Add Compound Measure Currents section and then press Scan Parameters Editor This opens the No
78. heck to prime the plenum with fluid from the Antibiotic source bottles instead of the Internal Buffer source bottle Adding Cells and Seal Test Hole Test Resuspend Cells before Dispense Performs a defined number of aspirate and dispense steps at the Cell boat position immediately before the cell transfer step Mix cycles When you have enabled cell resuspension select the number of aspirate and dispense mix cycles to use in the Resuspend Cell before Dispense option Time to Wait Before Seal Test Enter the number of seconds to wait between Prime Plate and Start Seal Test value range 1 1800 seconds Max mV Enter the maximum voltage applied to the cells in the seal test value range 995 mV to 1000 mV Min mV Enter the minimum voltage applied in the seal test value range 1000 mV to 995 mV Period ms Enter the duration for one cycle of the seal test value range 40 ms to 500 ms Set the limits in mV for the hole test Max mV Type in the maximum voltage value applied during the hole test Min mV Type in the minimum voltage value applied during the hole test Enter Remarks This field is reserved for entering comments Prompt on Run You are prompted to enter an experiment comment whenever the protocol is run when you start the experiment Enter Now Enables the field below where you can enter a comment to be appended to the data whenever the protocol is run 55 U
79. hoice 15 mL sterile conical centrifuge tubes Major laboratory supplier 119 User s Guide for lonWorks Quattro 120 To prepare and load the cells Step Action 1 Remove the media and rinse a T 75 flask of cells grown to 60 90 confluence with 5 mL of Versene at 37 C Add 3 mL of Versene and incubate flask at 37 C for 7 9 min or until the cells become rounded as viewed under a microscope Note Trypsin is not recommended as it may be detrimental to ion channel activity However some channels are not adversely affected by mild trypsin treatment We therefore recommend comparing trypsin containing media in side by side experiments to ensure that trypsin treatment is not detrimental to ion channel activity Tap cells loose and add 20 mL of serum free cell media to the flask Decant the cell suspension into two 15 mL conical centrifuge tubes and pellet the cells 50 x g for 4 min Add 2 5 3 5 mL or enough to achieve the desired final concentration of cells of serum free cell media and resuspend the cells carefully triturating them slowly with a p200 pipettor set at 170 UL Important It is important to resuspend the cells carefully and gently to avoid clumping and cell injury Note To ensure proper functioning of the system make sure that the final volume of cells is no less than 2 5 and no more than 3 5 mL Make sure there is a clean and dry v bottom reservoir in the Ce
80. ical pathway through the portion of the cell membrane that is resting on the small pores The associated electrode in the well allows you to clamp the membrane potential At this point the whole cell ion channel currents can be measured The IonWorks Quattro system integrates the PatchPlate consumable within a sophisticated network of fluidics and electronics systems The PatchPlate is a 384 well consumable for partitioning and positioning the cells Cells in suspension are added to individual wells of the PatchPlate by an integrated 12 channel or 48 channel pipettor A fluidics network comprised of peristaltic pumps solenoid valves and pressure regulators performs the PatchPlate priming fluid exchange and wash operations Electrical recordings of the ion channels are made with the 48 channel electronics head The experiment deck houses the PatchPlate plenum along with soak and wash stations cell and buffer reservoir positions and two compound plate stations 1 1 2 IonWorks Quattro System Components The main system components are listed below For a more complete description of the system components refer to Chapter 2 System Description Cabinet that contains the whole system Experiment deck Integrated 12 channel or 48 channel pipettor Integrated 48 channel electronics head E Head Fluidics system User s Guide for lonWorks Quattro Personal computer running IonWorks software that controls the instrume
81. ied A warning message will not be displayed until the Application is restarted The message will indicate the exact path of the folder but it will look like C Documents and Settings lt username gt Application Data Molecular Devices lonWorks V2 where lt username gt is the login of the current user The Application will display the warning at startup as long as any files are present in the temporary folder 4 3 3 Start Seal Test Tab This tab has options for selecting and preparing the PatchPlate for cell addition Hole and Seal Test configuration and allows the user to enter comments to be appended to the experiment Item Description PatchPlate Selection Select between a standard single hole or population PPC PatchPlate Prime Plate Select how much of the PatchPlate to use in the experiment Whole Plate All 384 wells are used 1 2 plate using Use rows A B E F for the first half and C D G H for the second half 1 4 plate using Use rows A and E for the first quarter B and F for the second quarter C and G for the third quarter and D and H for the fourth quarter 1 8 plate using Use every second well starting at column 1 in rows A and E for the first eighth then System Software Item Description the unused wells from rows A and E for the second eighth Continues similarly for rows B and F then C and G and D and H Add Perforation Agent during Prime Plate C
82. ill damage the vacuum pump b Place a new PatchPlate into the plenum cavity Operating the System Step Action c Seat the clamp frame on the PatchPlate station d Turn the PatchPlate vacuum switch from Release to Hold e Apply an even force to the clamp frame to create the vacuum seal Note The vacuum seal formation is complete when vacuum air leakage is no longer audible at the clamp frame interface 5 8 2 Preparing the Fluid Lines and 12 or 48 Channel Pipettor To prepare the 12 or 48 channel pipettor Step Action Clean and dry removable v bottom reservoirs Caution Ensure that you use the correct v bottom reservoir with the pipettor installed on your system The 12 channel v bottom reservoirs contain a single slot whereas the 48 channel v bottom reservoirs contain two slots The v bottom reservoirs are not cross compatible Insert one v bottom reservoir into the Buffer station on the experiment deck 2 Caution Check and verify that the removable reservoirs are fully seated in the appropriate station on the experiment deck prior to the start of the experiment Partial seating of the reservoir will damage the E head and or pipettor Fill the v bottom reservoir in the Buffer station with 5 mL of PBS or external buffer of choice Important Make sure that the solution dispensed into the reservoir is sterile and filtered through a 0 1 um filter 4 Close the access door and turn the
83. ime there is a conditioning train nor are interval sequence repetitions allowed with families Sequences of intervals within both the command voltage and conditioning trains can be repeated and in addition the whole of the conditioning train can be configured to repeat a set number of times General Layout The top half of the Voltage Protocol Editor has a graphical representation of the command voltage and or conditioning train as configured in the fields below This updates automatically as you change settings A View menu in the top left of the editor has options to view repetitions when these are configured and to view the sequence of stimuli delivered to each 12 head read bank individually or across all four read banks System Software You can print the graph from the editor s File menu The lower half of the dialog has three sets of configuration options selected by clicking on the Protocols buttons on the left Command Voltage Configure the pre signal and the command voltage Conditioning Train Enable and configure the conditioning train Scan Sequence Provides a summary of the entire sequence of the Voltage Protocol as well as o Set pre and post sequence durations and their holding level o Repeat conditioning trains o Enable read bank interleaving Voltage Protocol Editor Menus Commands in the File and View menus in the top left of the editor are described in the table Item Description
84. indicating failure to pass the seal test These are wells that failed to meet seal test thresholds during the pre signal step applied before each command voltage scan The failed wells from such a scan may be different from those that failed the dedicated seal test as some cells seal and others lose seal in the period between the scans 91 User s Guide for lonWorks Quattro 92 Color Description Default seal test criteria fail wells with less than 20 MQ or more than 1500 MQ resistance These thresholds can be changed from the Native Filter dialog opened from the Edit menu if a data file is being viewed Red Wells excluded from analysis by one of the user defined filters set in the Filters section below Yellow Hits A hit is a well whose metric meets the criterion set in the Summary section for the scan or scans selected in the same section If viewing the compound plate the average minimum or maximum value as selected in the Summary section from the replicates for that well is used Light Gray Wells excluded because manually rejected by the user Reject wells from the trace display area on the right of the data window Plate Legend and Statistics Area The Legend box in each Plate display has a legend for the color coding used and reports the number of wells under each category A separate box shows statistics for the metric currently configured taken across all the wells
85. ing trains for the first of the scans before and after the compound addition Sequence Repetitions Allows a subset of intervals in the train to be repeated The repeating intervals must be contiguous You can specify a delay between each repetition Repeated intervals are indicated by yellow shading in the graph Use Hide or Show Repetitions in the View menu to see the repetitions repeated or not 63 User s Guide for lonWorks Quattro 64 Scan Sequence The Scan Sequence view of the Voltage Protocol Editor provides an overview of stimulus configuration showing the entire stimulus sequence of pre signal conditioning train and command voltage including repetitions in the graph and reporting durations for the command voltage and conditioning trains as well as for the entire sequence The general holding potential is set here along with pre sequence and post sequence times There is also an option to repeat the entire conditioning train and you can enable read bank interleaving which delivers the pre signal and conditioning train to all 48 channels of the electronics head simultaneously after which the command voltage is delivered sequentially to each 12 channel read bank Use the Single Read Bank All Read Banks option in the View menu to change the graph to view the entire stimulus as it will be delivered to an individual read bank or to all four read banks this option is useful to see the difference made by t
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87. l Eate C A E ESE 141 vii Introduction Chapter 1 Introduction This chapter provides an overview of the lonWorks Quattro System This includes the principles of operation system components how the system operates and system specifications and requirements Additionally there is information regarding user and instrument safety Topic See Page Overview of the lonWorks Quattro System 2 lonWorks Quattro System Specifications and Requirements 5 Safety 7 Safety Alert Symbols 10 User s Guide for lonWorks Quattro 1 1 Overview of the IonWorks Quattro System The IonWorks Quattro system is a high throughput electrophysiology instrument that performs perforated patch voltage clamp recordings of ion channels Ion channels are protein molecules that span the lipid bi layer of the cell membrane They underlie many physiological processes such as neural signaling cardiovascular function immune cell activation and muscle contraction The gold standard measurement methodology for studying ion channels is the patch clamp technique The reason this technique is so powerful is the fact that you are able to directly measure the fast electrical currents of ion channel activity while simultaneously controlling the membrane potential of the cell Although very powerful the patch clamp technique is extremely labor intensive and has a limited measurement throughput The novel technology behind the lonWorks Quattro couples t
88. l Editor dialog Data Files Press New Metric or Edit Metric in the User Metric List Editor dialog opened with the main Edit menu User Metric List command In general one value is output per well and per scan for the settings made in this dialog An exception occurs for command voltage families which have multiple traces tied to a single scan label such as Pre compound Scan In addition this one value might be a combination of two or more intermediate values The value might be a combination of two measurements made within each scan e g the difference between the peaks at two different regions of the command voltage This is referred to as a Compound metric The value might be derived from within a family e g the mean metric value across the family This case would use the Cross Scan Reduction option to define an operation that combines the family values Even though there is one trace per member of the family only one value will be output per scan if the Cross Scan Reduction is used 81 User s Guide for lonWorks Quattro 82 The value might be a combination of metric values taken from subsequent scans within the experiment e g in a single compound addition experiment the value could be the difference between the metric measured on each scan and on the reference scan in this case always the pre compound scan Results for the pre compound scan will all then be zero i e the difference between the scan values
89. lls station Caution Check and verify that the reservoirs are fully seated in the experiment deck Partial seating of the reservoir will result in damage to the electronics and or pipettor Pour the cell suspension into the cell boat on the experiment deck Important Check and verify that the cell suspension is evenly distributed in the v bottom reservoir Due to fluid surface tension the cell suspension may collect at one end of the reservoir upon addition The cell suspension must be evenly distributed in the v bottom reservoir to ensure proper cell pipetting into the PatchPlate by all 12 or 48 channels of the pipettor Operating the System Step Action Install a clean and dry v bottom reservoir into the Buffer station on the experiment deck Caution Check and verify that the reservoirs are fully seated in the experiment deck Partial seating of the reservoir will result in damage to the electronics and or pipettor Dispense 5 mL of external buffer into the Buffer reservoir Close and secure the instrument cover 5 10 5 Loading a New PatchPlate Caution Keep the top surface of the plenum dry at all times The top surface of the plenum houses the vacuum port and the presence of moisture will damage the vacuum pump To load a new PatchPlate Step Action Check and if necessary unload the old PatchPlate from the PatchPlate station on the experiment deck a Turn the PatchPlate vacuum
90. lly button after accepting the System Software Item Description Scale All Axes settings Auto Scale Maximizes the X axis to show the full time course of the response and scales the Y axis to include the full response using the available graph area Manual Scale Enter the X and Y axis ranges to view Apply Scaling Globally Use after setting scale options in the Scale All Axes dialog to use the settings for all wells viewed Show Hide Difference When you have two scans on view set in the Define Data group in the Summary section you can view the difference between the two traces A dialog opens for you to select which trace to subtract from which This option is disabled for command voltage family scans This command applies to the current well only Graph Popup Menu The graph popup menu has a range of options described in the table Item Description Graph View Options A range of options allow you to change colors use data points configure grid line and axis appearance etc Note All these options can be accessed from the Customization dialog opened by double clicking on the graph Maximize This opens the graph into a new window that occupies the entire screen for a close look at the trace Export Dialog Opens the Exporting dialog to configure options to print the graph copy to the clipboard or save as a file See Export Trace Images page 103
91. ltage or subtract traces The graph popup menu has a wide range of settings to change the graph appearance These options can also be accessed from the Customization dialog if you double click on the graph You can open the graph into its own window or export it as a file by copying to the clipboard or printing Note You can get a slightly enlarged view of trace graphs in their own window by double clicking on a well in the Plate display The display in this view has the same options as the view in the right hand side of the data file Manual Reject A small checkbox at the bottom left of the trace graph allows you to manually reject that well excluding its response from analyses configured in the Summary section The PatchPlate well is colored light gray in the Plate section graphic and the traces in the trace graph are also grayed Toolbutton Commands There are four toolbuttons at the top of the trace display area described in the table Item Description Displays a second plot of the command voltage when a pre or post compound scan is in view or Command Voltage the hole test or seal test step when either of these Display responses is in view Note This setting persists as you change wells This button opens the Scale All Axes dialog to set axis ranges to view Set Plot Scale The dialog rescales just the current graph To apply the settings to all wells that you open press the Apply Scaling Globa
92. ltered out data are included in these statistics Define Data Select the scan or scans to measure If two scans are selected a predefined metric is applied to both and then the resulting two values per well are combined in order to give one output value per well Define Screen This section defines the single metric per well output by the summary Metric Select a predefined metric to be used It will be applied to the scan or scans selected in Define Data Define new metrics Edit User Metric List if the metric you want is not present Cross comparison The mathematical relationship used to compare the metric values from Scan A and Scan B if two scans are selected in Define Data The options are Difference 100 Scan B Scan A Scan A Absolute Value of Difference Difference Scan B Scan A Absolute Value of Difference Ratio Scan B Scan A Absolute Value of Ratio 97 User s Guide for lonWorks Quattro 98 Item Description Define hits Summary output values configured in the Define Screen section that fall in the range defined in this section are counted as hits Hit wells are colored yellow in the Plate display and the number of hits reported in summary statistics Discriminator Select Less than or equal to or Define Threshold Greater than Threshold Value The threshold metric value Note When determining a threshold value for use with
93. mbination of external and internal solutions This can be determined by performing a run with a particular solution pair with OmV input as the Liquid Junction Potential Correction After the run the value of the baseline current can be measured using user metrics and any voltage error can be determined using Ohms Law the baseline current value and the command voltage value The value input in the Liquid Junction Potential Correction field will be subtracted from the command voltage 5 5 Preparing the Perforation Agent The IonWorks Quattro system uses a perforation agent that forms low resistance pathways through the cell membrane for gaining access to the inside of the cell The low resistance access allows the passage of only small ions and thereby prevents the washout of cytosolic constituents Amphotericin B a polyene antibiotic is the recommended perforation agent for use in the lonWorks Quattro system 5 5 1 Using Amphotericin B Amphotericin B in powder form is dissolved in dimethylsulfoxide DMSO and diluted in the internal buffer Amphotericin B is a poorly soluble compound Therefore it is crucial to dissolve the Amphotericin B in DMSO before diluting the solution in the internal buffer A recommended method is to increase the surface area of the Amphotericin B in powder form when adding the DMSO The final Amphotericin B solution must be uniformly clear and devoid of any precipitates Note The final DMSO concentra
94. n may result in hazardous UV exposure 1 4 Safety Alert Symbols 1 4 1 Electrical Symbols This symbol indicates the ON position of the main power switch O This symbol indicates the OFF position of the main power switch This symbol indicates the ON OFF position of a push push main power switch This symbol indicates that a terminal may be connected to another instrument s signal ground reference This is not a protected ground terminal This symbol indicates that this is a protective grounding terminal that must be connected to earth ground before any other electrical connections are made to the instrument A terminal marked with this symbol either receives or delivers alternating current or voltage A terminal marked with this symbol can receive or supply an alternating and a direct current or voltage CAUTION This symbol indicates the presence of high voltage and warns the user to proceed with caution CAUTION This symbol alerts you to consult the manual for further information and to proceed with caution 10 Introduction 1 4 2 Nonelectrical Symbols The following is an illustrated glossary of all nonelectrical safety alert symbols found on Molecular Devices instruments CAUTION This symbol illustrates a heater hazard Proceed with caution when working around these areas to avoid being burned by hot components gt This symbol indicates that a laser is present inside the in
95. n the plenum can be circulated during the Pre compound scans for example The total time duration cannot be set shorter than System Software Item Description the end of the last Check Seals operation in the Obtain Access step All other operations are allowed to overlap with the Measure Currents operations Select a basic experiment type from the options then open dialogs from the Voltage Protocol Editor and Scan Parameters Editor buttons to configure these No Fluid Additions Repetitive Scans Take repeated measurements without adding anything to the cells Single Compound Addition First take a control measurement and then take a second measurement after addition of a test compound Add Compound Measure Currents Dual Compound Addition First take a control measurement then take a second measurement after a first compound addition from the compound plate position defined in the Scan Parameters tab page 71 then take a third measurement after a second compound addition Repetitive Scans with Single Addition Take multiple scans before and or after a single compound addition See the sections below for descriptions of the configuration options for these experiment types 4 3 5 Voltage Protocol Editor Use the Voltage Protocol Editor to configure the sequence of voltage steps and ramps that the electronics head delivers to the PatchPlate wells Open the editor with the Voltage Protocol Editor bu
96. nalysis Viewing the Data The IonWorks Quattro system software displays the electrophysiological recordings in a 48 cell format during an experiment run Typical Introduction experiments consist of multiple electrical scans per cell Data updates occur in real time The scaling or zoom parameters can be changed on individual traces during data acquisition Viewing the Analysis Upon completion of the experiment the primary analysis window displays a summary of the experiment data along with a color coded interactive view of either the PatchPlate or the compound plate Color coding based on native and user defined metrics provides a rapid visual overview of the experimental results for each well in the plate view Wells with viable cell recordings are readily distinguishable from unsuccessful wells Wells that exhibit characteristics of interest can be highlighted as hits for screening purposes Individual wells from the plate view can be selected to access the detailed data on a cell by cell basis in separate windows Data is displayed as either electrophysiological measurements over time time profile or single number metrics per individual well statistics format The primary analysis window provides analytical and statistical tools Post run analysis can be conducted by selecting combinations of scans metrics and thresholds Additionally there is the option of auto exporting the data for post run analysis In this case the
97. nd Description More detailed messages are reported when this option is checked Receive detailed messages Note Use this option only for instrument service purposes and not during regular operation as it affects the timing of instrument operations When clicked the Save As dialog box will open prompting you to name the file and determine the destination of the saved file The saved logging file will have a log file extension Start Logging When clicked the logging file will stop collecting log Stop Logging and messaging data 4 2 4 Experiment Menu Send commands to run experiments from the Experiment menu Note Most Experiment menu items are disabled unless the lonWorks Quattro is connected and powered on Item Description Run Starts the experimental protocol Stop Stops the experimental protocol System Software Item Description Opens the Monitor dialog box that displays status messages from the instrument and indicates the Monitor time remaining in the experiment The Trace Groups window can be opened from the Monitor dialog box to display the data in real time Set Pipetior Type Set the software for the 12 or 48 channel pipettor 4 2 5 Utilities Menu The Utilities menu has commands to control the instrument for specific maintenance operations Note Most Utilities menu items are disabled unless the lonWorks Quattro is connected and powered on Comman
98. nd of the trace The resistance value is determined using the default resistance metric If the Hole Test traces are displayed Show Annotations also indicates which holes were blocked Graph scaling is set in the Scale All Axes dialog opened from the Plot menu Slightly different scaling options are offered for the seal test and hole test traces as opposed to the Measure Currents data traces as shown in the table below Scale All Axes Dialog Item Description Auto Scale Adjusts the Y axis of each plot to fit the data range Seal Test and Hole Test Plot Scaling Fixed Scale with Auto Bias Adjust Plots all traces with the same Y axis range scaled to the greatest data range Auto Scale Adjusts the Y axis of each plot to fit the Measure Currents data range Plot Scaling Manual Scale Set the Y and X axis ranges for all graphs manually 47 User s Guide for lonWorks Quattro 48 In addition to these viewing options individual graphs containing four traces each can be maximized to take up the full screen extensively reformatted and exported see Export Trace Images page 103 These options are all available from the popup menu IonWorks Messages The IonWorks Messages dialog displays messages between the host computer and JonWorks hardware Messages can be saved in an ASCII log file Options in the lonWorks Messages dialog are described in the table below Comma
99. nds thereby reducing the effective concentration of the compound in solution which will result in an apparent decrease in observed potency We recommend making compound plates fresh for each run or use glass coated compound plates or glass vial plates instead of plastic to minimize compound adsorption Turn the PatchPlate vacuum switch to the Release position Important Leave an old PatchPlate in the plenum during the instrument idle times to prevent ambient debris from entering the plenum and plenum tubing lines Continue with the post run analysis from the primary experiment window See Exporting Metrics on page 101 for a procedure on how to export a data file 5 11 Exporting Data Data signals recorded during a run may be exported in two ways 123 User s Guide for lonWorks Quattro Right clicking on a signal chart of an individual well and selecting Export Dialog Selecting Export Traces from the File menu and exporting data from multiple wells at the same time In the first case the data may be exported in multiple formats and in the second case the data is exported in a tab delimited format suitable for importing into a spreadsheet application such as Microsoft Excel 5 11 1 Exporting Data for a Single Well To export data from a single well Step Action 1 Double click on the well of interest in either the pre or post compound plate This opens the data chart for that well
100. not excluded by virtue of the seal test or user defined filters Values reported are Mean Median Standard deviation Minimum value Maximum value System Software These values are also reported in the Statistics section the bottom section under the Plate display They can be copied from there to the clipboard When the compound plate is viewed a third box shows the metric value for each of the four replicates in the well where the cursor is positioned 4 4 3 Filters You can define up to five different filters to be applied to selected scans within the recorded data and rapidly apply these independently or in any combination Filters define metric ranges such that wells that meet the filter criteria are filtered out These wells are colored red in the Plate display These wells can be excluded from further analysis in the Summary section Note that filters use metrics applied to the data traces after any data modification currently enabled Trace data can be modified with Leak Subtraction Offset Current and Boxcar Filter all in the Data menu see Data Menu page 50 Configure Filters To configure a filter select a filter number and name the filter Then in the Define Data section select the scan or scans from which you will take measurements in a metric to use for the filter If you select one scan the filter works by rejecting wells that fail to meet a metric threshold value where the metric is meas
101. nt records and transfers acquired data to spreadsheet compatible files 1 1 3 Operating the IonWorks Quattro System PatchPlate Settings Experimental protocols can be designed to specify the desired PatchPlate coverage sequence of priming operations fluid handling modes electrical test and measurement parameters and user defined metrics The utility of the PatchPlate consumable can be optimized by selecting either whole one half one quarter or one eighth PatchPlate coverage The IonWorks Quattro system can be programmed for one two or no compound additions Experimental Parameters Settings Additionally there is the capability to configure the duration of the compound incubation and the types of compound plates A wide variety of command voltage protocols can be built in the Voltage Protocol Editor dialog including step and ramp pulses repetitive pulses and command voltage families Designing Analysis Metrics Lastly a set of post measurement analysis metrics can be defined as part of the experiment protocol Metrics provide the means of distilling information in cell measurements into a single number for each metric defined Through the Metrics Editor dialog box up to 20 simple or combined metrics can be defined These metrics apply user specified mathematical methods to individual traces to arrive at a single value for each metric Metrics are computed in real time during data acquisition and are used to perform post run data a
102. nt Summary window see below with protocol configuration details of the current data file Trace Groups Toolbutton Opens the Trace Groups View window see below to display signal traces from the current data file Messages and Logging Toolbutton gt Opens the lonWorks Messages dialog to display status messages from the instrument during operation Messages can be logged see below Active Protocol Summary The Active Protocol Summary window View Active Protocol Summary or toolbutton provides a concise summary of the currently loaded protocol including key configuration settings and the estimated duration of each of the major steps as well as of the entire experiment A graph displays the command voltage You can open the protocol to edit directly from the summary window with the Protocol command in the window s Edit menu Other options in the File menu in the window are 45 User s Guide for lonWorks Quattro 46 Save Protocol As to save a copy of the protocol under a new name or to a new location Print to print a copy of the summary as it appears in the window Exit to close the summary window Experiment Protocol Summary The Experiment Protocol Summary window View menu or toolbutton provides protocol summary information for the currently selected data file The information reported is the same as that provided for the currently loaded protocol in the Active Prot
103. nts Preparing compound plates Preparing the liquid delivery system Preparing the experiment deck Setting up the experiment protocol Completing the experiment setup 107 User s Guide for lonWorks Quattro 5 3 Preparing the Cells 5 3 1 Cell Densities The optimal cell density for running on the IonWorks Quattro system needs to be determined experimentally The recommended cell density range for running single hole and PPC experiments is 0 5 x 10 cells mL to 2 x 106 cells mL Important Higher cell densities may result in reduced potency measurements for some compounds e g hERG inhibitors While growth patterns vary widely for each cell line we recommend 60 to 95 confluence levels for optimum ion channel expression and therefore cells must be subcultured in advance to allow for 60 to 95 confluency on the day of the experiment The cells are loaded into v bottom removable reservoirs for pipetting into the PatchPlate The minimum required volume for the final cell suspension should be a minimum of 2 5 mL for systems equipped with a 12 channel pipettor and 5 mL for systems equipped with a 48 channel pipettor 5 3 2 Cell Preparation Requirements Cells are removed from their growth media and are suspended in balanced salt solutions for preparing the final cell suspension The final cell suspension that is pipetted into the PatchPlate should contain evenly distributed cells with no clumping Cell preparation techniques
104. o before scanning The options available here depend on PatchPlate usage set in Prime Plate on the Start Seal Test tab AAO All at Once compound is applied to the whole plate before scanning begins HAO Half at Once compound is applied to half the plate scanned then the second half and scanned QAO Quarter at Once compound is applied to each quarter of the plate and scanned successively EAO Eighth at Once compound is applied to each eighth of the plate and scanned successively AAO The longest average delay between compound addition and scanning but the shortest overall experiment time EAO has the smallest average delay between compound addition and scanning but the longest overall experiment time Measurement Timing vs Scan Type This summary table shows key durations within the experiment as well as overall experiment time at current settings Times are affected by the Compound Incubation Time Scan Type and Use Extra Aspirate and Use Singleton pipettor options Tip Refer to the Minimum Incubation Time in the summary table and use this value for the Compound Incubation Time in order to avoid being prompted with a warning message For scan modes other than EAO the Compound Incubation Time must be sufficiently long to allow one dispensing mixing and washing sequence of the pipettor otherwise a warning will appear and the incubation time will be reset automatically Solvent Wash Options Check to
105. ocol Summary window see above however there is no option to edit this information since it comes from a recorded file Like the Active Protocol Summary window the Experiment Protocol Summary lists experiment settings and gives the estimated durations of experiment stages and of the entire experiment The command voltage is displayed A copy of the protocol can be saved from the window s File menu and the summary can be printed from the same menu Trace Groups The Trace Groups View window View menu or toolbutton displays data traces from each PatchPlate well for the current data file The window also opens automatically when an experiment is running to display data traces as they are acquired You can manually update the display in this mode with the Refresh command in the window s Plot menu At any one time the window displays traces for the 48 wells in a selected group 1 to 8 from a particular stage of the experiment Hole Test Seal Test System Software Pre compound Trace 1 2 etc Post compound Trace 1 2 etc Select the trace type from the Scan list box in the top left of the window and the well group from the Group spinner Traces are displayed in twelve graphs within the window with four color coded traces per graph The Show Annotations command in the window s Plot menu toggles the cell resistance value for each pre and post compound trace in and out of view at the right hand e
106. oep erisia Ri a EEA e EEEE soxeven 126 5 13 Exporting Metrics sess ciiise esiotsa oree iesean seses a ii tiores Eea Ettir sni ooer i esaa dreina Tie Teaka 126 5 14 Importing a Metrics File into Microsoft Excel c ccccscesssssseseesseeeeteteteseseseenenenesesesnenens 127 5 14 1 Importing Metric Data FileSiisisrei iinei iiia arieni a 127 5 15 Shutting Down the System After Completing All Daily Experiments 04 128 5 15 1 Cleaning the Syste Merih a e ee aar RE aA Tae A EE EE Ee E iat R EREE 128 Chapter 6 M intenantE nsession irises tessere sideri ness sae eo ni Er es E SK ESA ER iE e 131 6 1 Daily Maintenance cira i n e e Aa EN EE E AENEAN 132 6 1 1 Startof Day imoni na A AEAEE E EE N ES 132 6 1 2 Priot to Each Ruma scscscstssiestiesnvesscssstaessdeanenscesued asvaoutscerssvessatsnsessencets oadeturerseeseversabpuests 133 6 1 SENG of the Daysie ain ia ai 133 6 1 4 PatchPlate Contamination neieiet a a a aan anaa aie 133 6 2 Rechloriding the E Head Probesse sisie sornisoii iori ee ia E 134 6 9 Detecting a Salt BAd gers ssiri tiroen Tie aaa Dies Tatoea iis eE TER E TAa thea oats 135 6 4 Testing the Pipettobr iniseti atistin ta ao iire E Ea A n E Eis 136 6 9 Washing the PIpettn sssini iniii a EE R E 137 6 6 Upgrading the Instrument Firmware ccccceesesesssneesescsesnenenesesseeececeeeeeenesesesesnenens 137 Appendix A Technical Assistance sersoosrssssnesssnsavavsnsscsseovenenssoenesonnenedocusnsosensaseecoyacpnonsises 139
107. ommand voltage protocol It can be used for example to open channels after compound addition to allow access to the compound and then return to the holding potential to rest the cell before the pre signal Conditioning trains are inserted after the pre signal Cell currents are not recorded during the conditioning train except for the last few milliseconds as needed to establish a signal baseline After the conditioning train before the command voltage starts the voltage is held at a level set specifically to follow the train for the same duration as the pre signal interval Conditioning trains cannot be run with a command voltage family Configuration of the conditioning train is similar to that of the command voltage allowing a maximum of 10 intervals within the train any sequence of which can be repeated In addition the entire conditioning train can be repeated set on the Scan Sequence view of the editor You can set the conditioning train to run prior to both pre compound addition and post compound addition scans or just with the post addition scan Item Description The number of intervals within the conditioning train is represented on the left of this section by the number of buttons numbered left to right Interval Sequence Conditioning Train Add intervals by clicking the button to the right Protocol and remove by clicking the button Click on an interval button to open the Interval Settings
108. ottom reservoir from the Buffer station on the experiment deck Discard the contents and clean the reservoir Perform the shut down procedure see Chap 3 129 Maintenance Chapter 6 Maintenance This appendix provides the information and procedures required for maintaining the lonWorks Quattro system in proper working condition Topic See Page Daily Maintenance 132 Rechloriding the E Head Probes 134 Detecting a Salt Bridge 135 Testing the Pipettor 136 Washing the Pipettor 137 Upgrading the Instrument Firmware 137 131 User s Guide for lonWorks Quattro 6 1 Daily Maintenance The procedures below are quick start procedures that should be followed when using the lonWorks Quattro system More detailed operating procedures are found in Chapter 4 and Chapter 5 6 1 1 Start of Day Daily maintenance tasks to perform at the start of the day Step Action 1 Install previously used PatchPlate 2 Rinse all bottles with deionized water to remove ethanol residue Fill all bottles with the appropriate fluid a External PBS or External buffer of choice b Internal Internal buffer of choice c Wash Ca Mg free PBS or Ca Mg free External buffer of choice d Antibiotic Internal solution e Buffer reservoir PBS or External buffer of choice 4 Run Flush and Rinse command Verify proper vacuum pressure low and hi vac Hi gt 23 on dial gauge 5 Lo 9
109. perate the IonWorks Quattro system and run an experiment Topic See Page Quattro System for an Experimental Run 107 Preparing the Cells 108 Preparing Buffer Solutions 109 Preparing the Perforation Agent 111 Preparing Compound Plates 112 Preparing the Fluidics System 114 Preparing the Experiment Deck 116 Setting Up the Experiment Protocol 118 Completing the Experiment Setup 118 Exporting Data 123 Importing a Data File into Microsoft Excel 125 Exporting Metrics 126 Importing a Metrics File into Microsoft Excel 127 Shutting Down the System After Completing All Daily Experiments 128 105 User s Guide for lonWorks Quattro 106 5 1 Experiment Operations Overview After issuing the start command for running an experiment the following operations are executed 1 The 12 or 48 channel pipettor aspirates the appropriate solution from the Buffer Reservoir station on the experiment deck and performs multiple dispenses into the wells of the PatchPlate The fluidics system primes the plenum or lower chamber under the PatchPlate The 48 channel electronics head E head records the electrical current flow through the holes at the bottom of the PatchPlate i e the Hole Test The pipettor adds cells from the Cell Suspension Reservoir to the PatchPlate At this point there is a waiting period user determined for cells to reach and to seal the holes at the bottom of the Patc
110. plated stainless steel probes of the E head must be coated periodically with a silver chloride layer Over time the repetitive electrical recordings will deplete the E head probes of this silver chloride coating and will cause an inevitable degradation in the performance of the E head To apply a silver chloride coating to the E head Step Action Perform the system start up procedure see Chapter 3 Starting Up and Shutting Down the System Note Make sure that the E head wash bottle is filled with proper washing buffer Open the instrument access door Remove and clean the flat bottom reservoir in the Soak station Install the flat bottom reservoir into the Soak position on the experiment deck Connect the grounding clamp onto the chassis Caution Do not install the v bottom removable reservoirs into the Soak station on the experiment deck The v bottom removable reservoirs are allocated for use either in the Cells or the Buffer stations on the experiment deck The v bottom reservoirs are designed for 4 accommodating the 12 and 48 channel pipettor The v bottom reservoirs do not accommodate the 48 channel E head Placement of a v bottom reservoir in the Soak station will damage the E head Caution Check and verify that the removable reservoirs are seated properly in the appropriate stations on the experiment deck prior to the start of the maintenance procedure Partial seating will damage the electronics
111. port file Because more than one value is produced in this case the metric cannot be used in configuring a Filter and individual member values cannot be displayed in the Metric Summary table Define Cross Scan For combination of metric values from different System Software Item Description Reduction scans check Add Cross Scan Reduction in the Define Type section then depending on the experiment type and whether or not a family is configured select a reference scan and the way the metrics from each scan will be combined with those of the reference scan Family Scans If a command voltage family is configured in the Voltage Protocol Editor and the Cross Scan Reduction option is used the mean minimum or maximum metric value across the family members can be determined Because a single value is produced by the metric in this case the result can be used in configuring a Filter and the metric value can be displayed in the Metric Summary table Alternatively select Reference Value for the Cross Comparison Method and a Family Member Number for the Reference Scan to output the metric for that family member in all the scans in the family This option allows a member value to be used in filtering data and generating statistical analyses of specific members Other Multiple Scan Experiments If a command voltage family is not configured select one of the scans within the experiment and set a comparison method
112. pounds that may adhere to the interior surface of the pipettor tips The pipettor aspirates a volume of compound equal to the volume to be transferred to the PatchPlate and then holds that volume for the time entered in the Duration field Expel to After holding the compound the pipettor can either Waste Expel the volume to waste in the pipettor wash station then return to the compound plate Compound Plate Mix the aspirated compound back into the compound plate Set the number of mix cycles in the Mix Cycles field In either case the pipettor then aspirates the transfer volume and moves to the PatchPlate to complete the compound transfer Note The location to perform prewetting is enabled only for Dual Addition experiments In Dual Addition exneriments the nrewettina sten can System Software Item Description only be performed at one location or the other not both The mix cycles option can be used to remix compound before transferring by setting Duration to zero Enter voltage offset correction values to be subtracted from pre compound and or post compound scans Liquid Junction Potential Correction 4 3 8 Scan Parameters Dual Compound Addition To configure scans with two compound additions select Dual Compound Addition in the experiment type options in the Add Compound Measure Currents section and then press Scan Parameters Editor This opens the Dual Addition Parameters dialo
113. rect damages including damages for loss of business profits business interruption loss of business information and the like arising out of the use or inability to use IonWorks even if MDC has been advised of the possibility of such damages Because some states do not allow the exclusion or limitation of liability for consequential or incidental damages the above may not apply to you MDC s liability to you for actual damages from any cause whatsoever and regardless of the form of the action whether in contract tort including negligence product liability or otherwise will be limited to 50 Contents Contents Chapter 1 Introduction sesssscscsssssssssssscsssssssscssscsssssssscsssssssessssssesssessesessssassssssesssssasssesesees 1 1 1 Overview of the lonWorks Quattro SySteM ccececccsceeeteteesesesneneesesesesneneneseseseeseseeeenenes 2 1 1 1 How the IonWorks Quattro System Works ccccecssscesseeteteeeceeeteneesesestsnenenenenesseees 2 1 1 2 IJonWorks Quattro System COMPponents ccecccecsesssesceseeseessseeseseeeneteesesesesnensenesssesees 3 1 1 3 Operating the JonWorks Quattro SySteM ccccsceeseeceesseteteesesesesneesesestsneneenesseeeees 4 1 2 IonWorks Quattro System Specifications and Requirements ss ssssssseesessesreeressesseresses 5 1 2 1 Electrical Requirements iire ree a n E esse EE e Eea Ea ESEE EEE R E iA 5 1 2 2 Physical Specifications and Requirements cccccccssssseseesssesceceeeseteteses
114. red simultaneously to all 48 probes after which the command voltage is delivered sequentially in banks of 12 as usual The electronics head is compatible with both single hole and multi hole PatchPlate consumables An automatic switching mechanism is built into the electronics and controlled by the system software Note There are no user serviceable parts on the amplifier boards Caution Do not remove the front panel covers as the boards are susceptible to damage from static discharge 2 6 PatchPlate Consumable The PatchPlate consumable is a 384 well platform for obtaining simultaneous parallel electrical recordings Two types of PatchPlate consumable can be used on the lonWorks Quattro system 23 User s Guide for lonWorks Quattro 24 Single hole version clear plastic the original consumable marked PatchPlate for lonWorks HT The multi hole population patch clamp version purple toned plastic marked PatchPlate PPC for lonWorks Quattro The PatchPlate is composed of an injection molded piece that defines the 384 wells attached to a bottom plate that forms the bottom of the wells and which contains the small pores on which cells are patched one pore per well in the original PatchPlate 64 pores per well in the PatchPlate PPC 2 6 1 Well Arrangement and Capacity The 384 wells of the PatchPlate are arranged in a grid of 8 rows by 48 columns The capacity of each well is 12 uL PatchPla
115. repetitions of the command voltage during each of which the voltage of this interval changes by the value set in the field above Acquisition Sets the data sampling period in milliseconds per sample Eight sampling rates are available 0 1 0 2 0 3 0 4 0 8 1 6 3 2 and 6 4 ms Pre signal Setup Sequence Repetitions Sets a voltage step for estimating the leak conductance prior to the main signal The pre signal step is compulsory Initial The voltage held for the first half of the pre signal in millivolts Final The voltage that is stepped to for the second half of the pre signal interval The Final potential must differ from the Initial potential by at least 10 mV Interval The overall duration of the pre signal The voltage is held at the Initial potential for the first half of the interval and at the Final potential for the second half Allows a subset of intervals in the voltage protocol to be repeated The repeating intervals must be contiguous You can specify a delay between each repetition Repeated intervals are indicated by yellow shading in the graph Use Hide or Show Repetitions in the View menu to see the repetitions repeated or not 61 User s Guide for lonWorks Quattro 62 Item Description Repetitions cannot be combined with the family option Conditioning Train A conditioning train is a voltage stimulus used to precondition the cell prior to applying the c
116. ric results file 5 14 Importing a Metrics File into Microsoft Excel Metric data from multiple wells of the PatchPlate or compound plate may be imported into a spreadsheet application such as Microsoft Excel Opening the data file within Microsoft Excel will launch the File Import Wizard which will guide you through the process 5 14 1 Importing Metric Data Files To import metric data files into Microsoft Excel Step Action 1 Open Microsoft Excel application and select Open from the File menu In the Open File dialog box navigate to the data file location Note Make sure that All Files in the Files of Type field is selected Select the metric file that you wish to open it will have a iha file extension and click Open This will launch the three step Import File Wizard Accept the default settings by clicking Next and Finish 127 User s Guide for lonWorks Quattro 128 5 15 Shutting Down the System After Completing All Daily Experiments When you are finished running all experiments for the day the system should be cleaned and powered down Important Cleaning the system should be done every day to avoid contamination problems 5 15 1 Cleaning the System To prepare the system for shut down Step Action 1 Wait for all instrument operations to complete and for the traffic light icon on the main window toolbar to turn green 2 Open
117. s Service Representative Step Action Check that the instrument power cord is connected to the room outlet with the specified requirements Note The lonWorks Quattro instrument connection for the power cord is located at the back of the power supply module inside the electronics cabinet Caution The instrument main power switch located on the front of the power supply module in the electronics cabinet must be in the OFF position prior to plugging in the power cord Check that the ethernet cable is connected between the lonWorks Quattro system and the computer Note The ethernet connection for the lonWorks Quattro is located at the back of the control module inside the electronics cabinet The ethernet connection for the computer is on the ethernet card receptacle in the back of the computer unit labeled Instrument Caution The computer contains another ethernet connection for networking purposes This ethernet connection is labeled Network Do not plug the instrument ethernet cable to the local area network LAN connection Check that the Plenum and Wash waste bottles are empty Check that the main vacuum line is connected to the proper port on the Wash waste bottle Check that the PatchPlate vacuum switch is set to the Release position Check that the instrument access door is closed and that the handle is turned to the lock position Turn on the vacuum pump or if the syst
118. s are part of the closed vacuum system and provide a reference point for setting the plenum differential pressure The height of the holding rack is set by the instrument manufacturer and should not be adjusted by the user Main vacuum input line Two position holding rack E Head F Head waste bottle Wash waste bottle Figure 5 Waste bottles System Description 2 10 System Computer The IonWorks Quattro system software runs on a computer with the following minimum configuration Intel Pentium 4 1 8 GHz or above 2 GB of RAM 150 GB hard disk drive 2 ethernet network interface cards 21 monitor at 1280 x 1024 resolution Windows XP Operating System 31 Starting Up and Shutting Down the System Chapter 3 Starting Up and Shutting Down the System This chapter provides the information and procedures required to properly start up and shut down the IonWorks Quattro System In order to ensure proper communication between the IonWorks Quattro system control software and hardware the start up and shut down procedures described in this chapter should be followed precisely Topic See Page Starting Up the lonWorks Quattro System 34 Shutting Down the lonWorks Quattro System 35 33 User s Guide for lonWorks Quattro 34 3 1 Starting Up the IonWorks Quattro System Prior to start up of the lonWorks Quattro system the instrument installation must be completed by a Molecular Device
119. ser s Guide for lonWorks Quattro 56 4 3 4 Access gt End Tab This tab has options for obtaining and testing electrical access to the cells and for configuring the type of experiment to run It has access to other dialogs to configure the voltage protocol and the plate scanning protocol Item Description Obtain Cell Access Set the sequence of operations up to 7 steps and their durations for the Obtain Access portion of the experiment Each step can be configured as one of the following step types Introduce Agent Introduce the Perforation Agent into the plenum This operation has a fixed duration of 70s Introduce Internal Add liquid from the Internal Buffer source This operation has a fixed duration of 70s Introduce Internal Add liquid from the Internal Buffer source This operation has a fixed duration of 70s Circulate Fluid Circulate the liquid in the plenum Circulation can be set to last from 1 to 1200 seconds Pause Pause the instrument for 1 to 1200 seconds Check Seals Run an additional Seal Test This operation has a fixed duration of 50 s Null Operation Omit the step Force total time of Obtain Cell Access steps to You can set the total time of the Obtain Access steps of the experiment when this option is checked The Obtain Access experiment steps are then allowed to overlap with the current measurements in the Measure Currents step Using this option the fluid i
120. ser interface appear below Following sections deal more closely with parts of the interface that require deeper explanation System Software 4 1 1 Title Bar The Title Bar extends across the top of the main window It displays the name of the application followed by the name of the data file open in the program or the most recently accessed data file if more than one is open at a time The right hand end of the Title Bar has standard Windows minimize maximize and resize buttons 4 1 2 Menu Bar The Menu Bar lies below the title bar It groups related lonWorks commands in menus See Menu Bar Commands below for descriptions of the menus and the commands they contain 4 1 3 Toolbar The toolbar lies below the Menu bar It contains toolbuttons for commonly used commands The toolbar can be toggled in and out of view with the View menu Toolbar command The buttons on the toolbar are as follows Button Name Description Opens the Open lonWorks Data File Open Data File dialog to locate and open a data file ihe Saves the data file currently showing Save Data File on screen Only enabled if settings for the file have been changed Load Active Protocol Opens a saved lonWorks protocol file from File ihp Save Active Protocol Saves the active protocol to disk 39 User s Guide for lonWorks Quattro 40 Name Description Edit Active Protocol Opens the Protocol Editor allowing chang
121. shields against electrostatic discharge ESD to the enclosed circuit boards but individual probes remain extremely susceptible to damage caused by ESD Note The electronics head performance matches the high fidelity measurement levels demanded of industry standard patch clamp systems 2 5 1 Electronics Head Mechanism of Action The Amplifier System 48 high gain trans impedance amplifiers arranged in four banks of 12 are enclosed in the electronics head housing These amplifiers perform the System Description same basic current to voltage conversion as the head stage in a classical patch clamp apparatus All operations on the IonWorks Quattro system are performed in a voltage clamp mode Command and hold voltages are specified in the software and generated by a digital to analog converter in the control module Output signals from the amplifiers are multiplexed onto a cable in groups of 12 and returned to the control module where they are digitized Acquisition occurs when the command voltage is delivered Other parts of the stimulus i e conditioning trains optional are not recorded All probes are maintained at user specified holding voltages between configured steps During a read the user specified waveform including conditioning trains and command voltage is generally applied to one bank of 12 probes at a time until all four banks have been read With the read bank interleaving option the conditioning train is delive
122. spense is used with Use Extra Aspirate the extra aspirate volume is drawn with each transfer not divided among all four singleton transfers Singleton Dispense adds roughly 2 5 minutes to the total running time of a PatchPlate shown in the Measurement Timing vs Scan Type table Pipettor Prewetting Option Enable for use with sticky compounds that may adhere to the interior surface of the pipettor tips The pipettor aspirates a volume of compound equal to the volume to be transferred to the PatchPlate and then holds that volume for the time entered in the Duration field Expel to After holding the compound the pipettor can either Waste Expel the volume to waste in the pipettor wash station then return to the compound plate Compound Plate Mix the asnirated comnound System Software Item Description back into the compound plate Set the number of mix cycles in the Mix Cycles field Location Select the plate on which to apply prewetting only one compound addition can use prewetting After prewetting the pipettor aspirates the transfer volume and moves to the PatchPlate to complete the compound transfer Note The mix cycles option can be used to remix compound before transferring by setting Duration to zero Liquid Junction Enter voltage offset correction values to be subtracted Potential Correction from pre compound and or post compound scans 4 3 9 Scan Parameters Repetitive S
123. strument 11 System Description Chapter 2 System Description This chapter provides detailed information about the lonWorks Quattro system components Topic See Page lonWorks Quattro System Components 14 System Cabinet 15 Experiment Deck 16 12 or 48 Channel Pipettor 21 48 Channel Electronics Head 22 PatchPlate Consumable 23 Electronics Cabinet 25 Fluidics System 26 Source and Waste Bottle System 28 System Computer 31 13 User s Guide for lonWorks Quattro 2 1 IonWorks Quattro System Components The IonWorks Quattro system is comprised of the following components System cabinet Experiment deck Integrated 12 or 48 channel pipettor Integrated 48 channel electronics head PatchPlate consumable Electronics Fluidics system Source and waste containers Personal computer running custom software that controls the instrument parameters records data and transfers acquired data to spreadsheet compatible files See Figure 1 for a picture of the Molecular Devices IonWorks Quattro system with the major components labeled 14 System Description Hin or Source bottles and nged aloes da holding rack a System cabinet Experiment stage 12 channel fluidics head and 48 channel electronics head aste bottles and holding rack Electronics compartments Fluidics panel Power switch and indicator Figure 1 The IonWorks Quattro
124. switch to the Release position b Remove the clamp frame c Remove the PatchPlate from the PatchPlate plenum Check and if necessary dry any traces of fluid on the top surface of the PatchPlate plenum Place the new PatchPlate into the plenum cavity Seat the clamp frame on the PatchPlate station Turn the PatchPlate vacuum switch from the Release to the Hold position Apply an even force to the clamping frame to create a vacuum seal Note The vacuum seal is complete when vacuum air leakage is no longer audible at the clamp frame interface 121 User s Guide for lonWorks Quattro 122 5 10 6 Starting the Experiment To start the experiment Step Action Close the instrument access door and turn the handle to the lock 1 a position Wait for the traffic light icon on the main window toolbar of the system software to turn green Click the green lighted button or select Start from the Experiment menu to start the experiment run 5 10 7 Viewing the Experiment Data After starting the run by clicking the green button in the main window toolbar or selecting Run from the Experiment menu the Trace Groups window will open and display the signals in real time while the software is recording and saving the data The experiment segment being recorded is shown in the Scan field and the current group of wells for which the signal is being recorded is displayed in the Group field
125. t and buffer boat if desired Make sure there is wash solution in the F wash bottle Select Wash Pipettor from the Utilities menu and check the boxes to wash the pipettor at the desired locations Select the number of washes 2 to do at each location each wash is four aspirate dispense cycles The order of the locations is fixed solvent boat followed by buffer boat followed by wash station Click OK to start the wash sequence The Utility running message is 3 displayed until the wash sequence is completed and the pipettor has returned to the park position 6 6 Upgrading the Instrument Firmware The Firmware Upgrade utility allows you to install new versions of the instrument firmware without requiring a field service call In most cases firmware updates will accompany updates of the Host application as well This procedure describes both steps Note this utility can only be used to update an instrument that is currently running version 2 0 Firmware Upgrading to v2 0 from an earlier version requires a fieldservice call 137 User s Guide for lonWorks Quattro 138 How to install software updates Step Action Uninstall the previous version of lonWorks V2 application Make sure you are logged in to the instrument using an administrator s account If you go to the Control Panel and select Add Remove Programs and then click on lonWorks V2 you should see buttons labeled Change and Remo
126. ta Tab page 79 for general description of the dialog which is the same as the Protocol Editor tab of that name and User Metric List page 81 for a description of the User Metric Editor dialog opened by clicking on the Edit Metric button Note This command only appears when a data file is open Native Filters Use the Native Filter Editor to change the resistance levels used to define No Seal wells in the current data file PatchPlate Seals When data files are recorded they always use the default definition of No Seal i e less than 20 MQ and greater than 1500 MQ These settings can be changed for a given data file e g to increase the data available in the plate PatchPlate Holes Resistance thresholds used to define blocked cells in the Hole Test are reported but cannot be adjusted A PatchPlate well is considered blocked if the resistance is less than 1 MQ or greater than 10 MQ during the Hole Test 4 2 3 View Menu System Software A description of the View menu items is listed below Item Description Toolbar When checked displays the main window toolbar Status Bar When checked displays the status bar Active Protocol Summary Toolbutton Opens the Active Protocol Summary window see below which reports configuration details of the currently loaded protocol reported in the Status Bar Experiment Protocol Summary Toolbutton Opens the Experime
127. tage stimulus value range 1 s to 1200 s Note The incubation time does not start until after the addition and mixing of the compound The Minimum Incubation Time includes the mixing time and the Maximum Incubation Time includes mixing time and in some cases washing time depending on the scan mode Select Scan Type Select how much of the PatchPlate to apply compound to before scanning The options available here depend on PatchPlate usage set in Prime Plate on the Start Seal Test tab AAO All at Once compound is applied to the whole plate before scanning begins HAO Half at Once compound is applied to half the plate scanned then the second half and scanned QAO Quarter at Once compound is applied to each quarter of the plate and scanned successively EAO Eighth at Once compound is applied to each eighth of the plate and scanned successively Note In this mode washing of the pipettor does not occur until after the Post compound scan is completed This behavior is intentional to minimize the delay between the compound addition and the measurement AAO The longest average delay between compound addition and scanning but the shortest overall experiment time EAO has the smallest average delay between compound addition and scanning but the longest overall experiment time Measurement Timing vs Scan Type This summary table shows key durations within the experiment as well as overall experiment time
128. te wells are identified as follows The 8 rows are labeled A H The 48 columns are labeled 1 48 Individual wells therefore are identified by a row letter and column number When the PatchPlate is loaded onto the experiment deck the well closest to the front left of the system cabinet is A1 The rear right well in this position is H48 the 384th well see Figure 3 System Description ABCDEFGH 48 PatchPlate uoe S ysSeM Pe8H F UdHTEIS ysem JOWEdIq Front of cabinet Figure 3 PatchPlate showing well locations 2 6 2 PatchPlate Packaging The PatchPlate consumables are packaged in groups of five The clear plastic packaging is heat sealed to an opaque plastic lid with perforations that allow removal of one PatchPlate at a time Each PatchPlate box contains 50 PatchPlates 10 x 5 packs The PatchPlates must be stored in a cool dry place The PatchPlate performance is assured for six months from the date of receipt 2 7 Electronics Cabinet Located at the lower right compartment of the system cabinet the electronics cabinet houses three modules Power supply 25 User s Guide for lonWorks Quattro Control Motor drive 2 7 1 Power Supply Module The power supply module has the main system power button on it This module supplies all the electrical power required for the IonWorks Quattro system Note The power supply module is configured to the end user s voltage set
129. tem used in an experiment The 96 well subset consists of every other well in a 384 well compound plate that is specified through the system software In PPC mode besides running the compound in quadruplicate as in single hole mode a 384 well compound plate can also be run in duplicate 1 compound well to 2 PatchPlate wells or single 1 compound well to 1 PatchPlate well In duplicate the pipettor can either take the first half of the plate row A H or second half of the plate row I P specified in the software Quantity of Fluid Dispensed The volume of fluid dispensed into the PatchPlate well from the compound plate is set in the Scan Parameters dialog opened from the Experiment Protocol Properties dialog You must select from a range of volumes offered in the dialog When one of these is selected the volumes of buffer and cells solutions added to the PatchPlate wells are automatically determined to give a specific known dilution factor see below The required fluid volume in each well of the compound plate must also account for the dead volume due to pipetting heights For example the dead volume for a 1 2 area 96 well plate Costar P N 3696 is approximately 35 uL So for example if using 3 5 uL aliquots with quadruplicate dispensing mode a minimum fluid volume of 49 uL 4 x 3 5 uL 35 uL should be added to each well of the 96 well plate 5 6 4 Concentration of Compounds in the Plate When compounds are added
130. tem Software Item Description Compound Define two separate measurements from one or two regions and combine these e g as a ratio Use Cross Scan Option Enable combination of metric values from different scans Configure this option in the Define Cross Scan section below Define Reduction Define one or two reductions depending on the metric type selected Simple or Compound If defining two reductions select a method to combine the values from the two reductions resulting in one output value Select the 1 Reduction or 2 Reduction option to view or edit each reduction in the other fields in the section Reduction Select a particular value to measure or calculate from the region defined immediately below in the dialog Available reductions are Absolute Value of Min The absolute value of the minimum current measured e g a minimum data point 4 nA is reported as 4 as is a minimum value of 4 nA Minimum Signal Time The time at which the minimum current is measured Absolute Value of Max The absolute value of the maximum current measured Mean The mean of the values measured within the search region a Minimum The minimum current value measured Maximum The maximum current value measured Maximum Signal Time The time at which the maximum peak current is measured Standard Deviation The standard deviation of the values measured within the search region
131. the instrument access door 3 Remove all removable reservoirs and compound plates i e Soak Plate 1 Cells Buffer and Plate 2 4 Discard the contents and clean all removable reservoirs Install clean and dry v bottom reservoir into the Buffer station on the experiment deck 5 Caution Check to verify that the v bottom reservoir is properly seated in the appropriate station on the experiment deck Partial seating of the reservoir will damage the electronics and or pipettor 6 Dispense at least 5 mL of 50 EtOH into the Buffer reservoir Drain all source bottles 7 O Important Turn the bottle to unscrew the bottle cap when connecting or removing the bottles All bottle caps are connected to internal tubing lines that will kink if bottle caps are turned Rinse all source bottles thoroughly with clean filtered water Fill each source bottles with 200 mL 50 EtOH Load all source bottles 10 Important Check that the labeling on the bottle cap matches the labeling on the annronriate source bottle The hottle can contains a tubina line that Operating the System Step Action connects to the appropriate station The bottle cap is distinguished by a label that corresponds to its matching bottle Installing the incorrect bottle cap to the source bottle will result in flow of incorrect solutions to various stations Turn the bottles when connecting or removing the bottles form the bottle caps Turning the bottle c
132. the summary be aware that the values displayed in the Plate Legend and Statistics Area are rounded to three decimal places whereas the values used for hit definition are much higher precision Values greater than three decimal places can be specified for the hits threshold Enabled when the compound plate is selected in the Plate display use this field to set how to combine replicate summary values for each compound plate well for the purpose of hit definition Options are Replicates Average a Minimum Maximum 4 3 4 Statistics The Statistics section provides an overview of results for all of the summaries Summary section defined in the file one row per summary The entire contents of the section can be easily copied for transfer into third party applications Select the summary to view in the Plate and Trace displays by clicking on its row in the Statistics area System Software The parameters reported in Statistics are the same as those displayed in the Legend and Statistics boxes in the Plate display however statistics for all defined summaries are always shown here Values reported are Number of blocked wells Number of wells with no seal Number of wells that are filtered out zero if Apply to Filtered Data in the Summary section is disabled Number of hits Mean of the summary values across the PatchPlate including or excluding filtered out results depending on Apply to Filtered Data setting
133. ting Data for a Single Fluidics System scsesee 26 Help Menu cceccceceeseesees 51 Internal Buffer c 110 IonWorks Messages 48 Loading a PatchPlate 116 Maintenance cccceeeeeees 131 Metrics 004 4 40 43 44 80 101 102 105 123 126 127 Microsoft Excel 105 124 125 126 127 PatchPlate Consumable 23 Perforation Agent 0 111 Pipettorssscccseskeliseleettiess 21 Pipettor Mechanism of ACTION oeeeeecseeeeeseeeeeeeeseees 21 Pipettor Wash Station 19 Perforated Patch Preparing and Loading the Gelsin 119 Preparing Buffer Solutions 109 Preparing Compound Plates cei ohiren 112 Preparing the Cells 108 Index 141 Preparing the Experiment Preparing the Fluid Lines and 12 or 48 Channel PIPettOL seierherrer 117 Preparing the Fluidics SYSUOM esceseseeseseereseeneseenens 114 Preparing the Perforation A BENT 111 Protocol Editor Dialog Protocols ee Rechloriding 134 Removable Reservoir 17 Salty discs cesssassaetesteteetescsonetensansest 7 Salt Bridge Scan Sequence ececeeeeeeeee 64 Shutting Down the System 128 Solvent Boat 142 Starting Up and Shutting Down the System Statistics hesi iii System Computer ccceeees System Description System Software System Specifications
134. tings 110 VAC 4 A or 220 VAC 2 A prior to shipment from the factory 2 7 2 Control Module This module is the central location for coordinating system operations The control module houses the IonWorks Quattro embedded computer system along with digital and analog cards for various input output functions It accepts the external ethernet line as well as the internal connections for the electronics head fluidic valves vacuum regulator and motion controllers 2 7 3 Motor Drive Module This module coordinates the electronics head and the pipettor motion operations as well as the fluidics pump actions The motor drive module houses a central motor interface board that interacts with the enclosed stepper motor and peristaltic pump drives It operates the system safety interlock for the access window along with connections for the various motor and pump drives and encoders 2 8 Fluidics System Located at the lower left compartment of the system cabinet the fluidics system performs priming fluid exchange and wash operations The fluidics system is comprised of a network of interconnected tubing with inline 26 System Description peristaltic pumps solenoid valves and a vacuum regulator There are three integrated peristaltic pumps Fluidics wash Electronics wash Plenum 2 8 1 Fluid Control As the pumps circulate the solutions in separate tubing pathways a series of inline solenoid pinch valves control the direction of
135. tion should not exceed 0 5 111 User s Guide for lonWorks Quattro The recommended method for use of Amphotericin B is to prepare the solution fresh each time prior to the start of an experiment 5 6 Preparing Compound Plates 5 6 1 When to Prepare the Compound Plate Depending on its complexity preparing the compound plate can take a variable amount of time For this reason it is best to plan your experiment carefully to ensure that the compound plate will be ready for use as soon as the cells are ready to be assayed 5 6 2 Compound Plate Types Either 96 or 384 well microplates can be used in the compound plate positions of the experiment deck in the lonWorks Quattro system You must specify the plate type from a list provided in the system software prior to instrument operation 5 6 3 Dispensing from Compound Plates IonWorks Quattro treats 96 well plates the same way in single hole and PPC modes 384 well plates however are treated differently in the different modes 96 Well Plate In both single hole and PPC modes the 96 well compound plate is mapped to the 384 well PatchPlate such that there are four corresponding PatchPlate wells for each compound plate well 384 Well Plate In single hole mode compounds are still run in quadruplicates on the PatchPlate A 384 well compound plate is regarded as four separate 96 well compound plates where a 96 well subset of a 384 well compound plate is 112 Operating the Sys
136. to calculate metrics relative to that scan in which case the reference scan is compared to itself giving uninteresting results in such a case For single compound addition experiments the reference scan is the pre compound scan by default For other experiment types you can select from amongst all the scans Methods of comparison are the same as those offered for compound reductions in the Define Reduction section 85 User s Guide for lonWorks Quattro 86 4 4 Data Files IonWorks data files ihe open into windows in the main work area of the interface The data file windows have a graphical display area on the right hand side to view traces from individual PatchPlate or compound plate wells Below the graph or graphs is a summary of metrics for the well or wells displayed above On the left hand side of the window are four expandable sections for configuring and viewing results The sections are Plate Displays a grid representing the 384 well PatchPlate or the compound plate Wells in the grid are color coded to represent their status with respect to filters and Summary metrics Filters Allows configuration of up to five different filters using metrics defined in the file to exclude data from analysis Summary Apply metrics and set thresholds to define hits shown in the plate display above Any number of hit definitions can be created Only the currently selected Summary defines the well hit highlighting
137. to the PatchPlate they are diluted in the buffer and cell solutions already in the PatchPlate wells The amount of dilution depends on the relative volumes of each when you select the volume of compound to add the lonWorks automatically calculates volumes of buffer and cells solution so that the compound will be diluted by a simple ratio such as 1 3 or 1 2 Ensure that you take these dilution factors into account when you develop your assay and prepare compound plates 113 User s Guide for lonWorks Quattro Volumes and dilution for single compound additions are listed in the following table Compound Buffer Cells Dilution Total Vol 3 0 3 0 3 0 3x 9 0 3 5 3 5 3 5 3x 10 5 4 0 2 0 2 0 2x 8 0 5 0 2 5 2 5 2x 10 0 6 0 3 0 3 0 2x 12 0 Volumes and dilutions for dual compound additions are listed below 5 7 Preparing the Fluidics System Note When starting an experiment the source containers should be clean refer to Shutting Down the System After Completing All Daily Experiments on page 128 for a procedure to clean the system after an experiment To prepare the liquid delivery system Compound Buffer Cells Dilution Total Vol 2 0 6 0 2 0 2 0 3x 2x 12 0 2 5 2 5 2 5 2 5 3x 4x 10 0 2 5 3 7 2 5 2 5 3x 3x 11 2 4 0 4 0 2 0 2 0 2x 3x 12 0 2 0 6 0 2 0 2 0 3x 2x 12 0 Step Action 1 Perform the system start up procedure Drain all waste bo
138. tomatic Trace Export on the Analyze Export Data tab in the Protocol Editor dialog Or export metrics from a saved data file Export Traces command in the File menu The same configuration dialog is used for both protocol and data file export System Software Section Description PatchPlate Wells Labels the exported data using PatchPlate row and column designation Each PatchPlate well is exported in a separate file Export By Compound Plate Wells Labels the exported data using compound plate row and column designation Each file contains the data from the four replicates wells of the PatchPlate that correspond to the Compound Plate wells being exported Wells to Export All in Plate Exports the data for all wells Along Row Allows you to export data from defined rows using the Start from and End with fields below Along Column Allows you to export data from defined columns using the Start From and End With fields Data Sets to Export Select the types of trace recordings to export Options are the Hole Test Seal Test Pre and Post Compound Scans and the Command Voltage Other types of traces may appear in the list depending on the experiment type such as Scan 1 etc for a Fixed Scan experiment Use the Shift or Control buttons to select multiple trace types Export File Prefix Enter a file name prefix for the exported files The well number for each file is automatically appended along
139. ttles Important Turn the bottle to unscrew the bottle cap when connecting or removing the bottles All bottle caps are connected to internal tubing lines and turnina the bottle cans instead of the bottles will introduce kinks into the 114 Operating the System Step Action internal tubing lines Check that all bottle caps are tightly secured prior to the start of an experiment The waste bottle caps are connected to the main system vacuum line and must be tightened to create a closed vacuum system Loose bottle caps will result in vacuum leaks and improper priming of the PatchPlate Avoid exerting excess force in tightening the bottle caps as over tightening can damage the locking threads on both the cap and the bottle Warning Always check the fluid volume in the waste bottles prior to starting an experiment to avoid overflowing and saturating the vacuum systems The waste bottle caps are connected to the main system vacuum line Overflow of the waste bottles will damage the vacuum system Drain all source bottles O Important Turn the bottle to unscrew the bottle cap when connecting or removing the bottles All bottle caps are connected to internal tubing lines and turning the bottle caps instead of the bottles will introduce kinks into the internal tubing lines Rinse source bottles thoroughly with clean filtered water Prepare 5 mg amphotericin in 180 uL DMSO sonicate for 5 min add
140. tton on the AccessEnd tab in the Protocol Editor dialog Data acquisition occurs only during delivery of the command voltage Prior to delivery of the command voltage you must configure a voltage step of at least 10 mV to measure resistance in the pre signal Additionally you can 57 User s Guide for lonWorks Quattro 58 configure a conditioning train to run prior to the command voltage following the pre signal Under normal operation the sequence of pre signal conditioning train and command is delivered to each bank of 12 in the electronics head one after the other A read bank interleaving option however delivers the pre signal and conditioning train to all 48 heads at once after which the command voltage is delivered sequentially to the four 12 head banks The command voltage and conditioning train can each be configured with up to 10 intervals Intervals can be defined as steps of constant voltage or voltage ramps One step interval within the command voltage can have a family of voltages configured defined with a regular positive or negative shift in the specified voltage in that interval for a given number of steps Under these conditions the command voltage is repeated as many times as there are family steps configured with the command voltage changing in that interval as defined for each subsequent iteration of the sequence Note A command voltage family cannot be configured at the same t
141. ual memory warnings on the host PC with insufficient RAM 2 GB RAM is recommended for v2 0 Therefore it is recommended that the user not open any other data files or programs on the host PC until the experiment is completed Item Description Select Plate Types Select a plate type for the compound plate and for the solvent plate if using enable a solvent plate at Solvent Wash Options section Set which plate 1 or 2 is the compound plate in the Compound Location field in the next section The Plate Type list has predefined compound plates used with the lonWorks Quattro system Contact Molecular Devices to request additional plate definitions 384 Well Plate Settings When a 384 well plate is selected as the compound plate select the starting well A1 A2 B1 or B2 to define the quadrant from which to commence pipetting See supplemental Compound Plate Maps PDFs located in the Help menu Compound Locations and Dispense Volumes Compound Location Select plate 1 or 2 as the compound plate If you have the other plate location selected for solvent its location automatically changes if you change this setting The selected plate locations are reported at the top of the dialog beside the graphic Volume of Compound Select the volume of compound to be dispensed 3 0 3 5 4 0 5 0 or 6 0 microliters Volumes of buffer solution and cell solution are automatically adjusted depending on this setting to
142. upplemental Compound Plate Maps PDFs located in the Help menu Compound Locations and Dispense Volumes Compound Location Set the order the plates are used The selected plate locations are reported at the top of the dialog beside the graphic Volume of Compound Select the volume of compound to be dispensed Four different combinations of 1 and 2 compound volumes are permitted resulting in specific dilution ratios for each of the compounds These options are summarized in the table below the default setting is bolded Compound Buffer Cells Dilution Vig 2 0 6 0 2 0 2 0 3x 2x 12 0 2 5 2 5 2 5 2 5 3x 4x 10 0 2 5 3 7 2 5 2 5 3x 3x 11 2 4 0 4 0 2 0 2 0 2x 3x 12 0 Buffer and cell volumes are reported at the bottom of the section in the dialog Select Timing and Scan Parameters 1 amp 2 Compound Incubation Times Enter the length of time that you want the cells incubated with compound for each of the compound additions before applying the voltage stimulus value range 1 s to 1200 s Note that the incubation time does not start until after the addition and mixing of the compound The Minimum Incubation Time includes the mixing time and the Maximum Incubation Time includes mixing time and in some cases washing time depending on the scan mode Select Scan Type Select how much of the System Software Item Description PatchPlate to apply compound t
143. ured from that scan If you select two scans you will need to select a method to compare the two metric values measured in each scan and set the threshold in terms of the single comparison value Having selected the scans you will take metrics from select the metric in the Define Screen section If the metric that you want is not currently defined use the User Metric List Editor dialog Edit menu or toolbutton to define a new metric Where the filter uses the comparison between two scans select the method to compare the metrics 93 User s Guide for lonWorks Quattro Finally set the threshold value for the metric one scan or comparison value two scans Wells that fail to meet this threshold are filtered out when you enable the filter Further explication of the commands involved is given in the table Item Description Select the number of the filter 1 to 5 When a number is selected the remainder of the fields in the Filters section are populated with Number settings for that filter Undefined filters default to the Resistance metric on the hole test scan with a threshold of less than or equal to zero Name The name of the current filter Select the scan or scans to measure to define the filter Select the segment of the experiment to which the metrics will be applied The scans to choose from will always include the Hole Test and Seal Test but depending on the type of experiment there may be
144. uses both extracellular i e External Buffer and intracellular solutions i e Internal Buffer for maintaining and optimizing the electrochemical gradient of the cell during electrophysiological recordings 5 4 1 External Buffer The external buffer is equivalent to the bath solution in a patch clamp configuration External buffer has the following applications in the IonWorks Quattro system Preparation of cell suspension Filling the PatchPlate The recommended external buffer for use in lonWorks Quattro system is a balanced salts solution e g Dulbecco s Phosphate Buffered Saline solution 1X with calcium and magnesium filtered with a 0 1 um filter External solutions used in conventional patch clamp electrophysiology may also be used on IonWorks e g HEPES buffered balanced salts solution 109 User s Guide for lonWorks Quattro 110 Important Any solution used for pipetting into the PatchPlate must be both sterile e g 0 1 um filtered 5 4 2 Internal Buffer The internal buffer is equivalent to the pipette solution in a patch clamp configuration Internal buffer has the following applications in the IonWorks Quattro system Priming the plenum at the beginning of the experiment Dissolving the perforation agent The choice and the composition of the internal buffer depend both on the host cell and the ion channel of interest Note While internal solutions may be optimized for t
145. vacuum switch is located on the main platform of the IonWorks Quattro system immediately next to the plenum It has two positions RELEASE and HOLD Two source bottles serve the PatchPlate station Internal Buffer is the main source bottle Fluid exchange operations such as introduction of perforating agent use the Antibiotic source bottle The waste bottle is labeled Plenum Waste 2 3 5 Pipettor Wash Station The pipettor wash station is an embedded reservoir with 12 or 48 wash channels for individually cleaning the pipettor needles A peristaltic pump brings wash solution from the F Head Wash source bottle while a solenoid pinch valve connected to the system vacuum line opens an exit drain connected to the Wash Waste bottle 2 3 6 Compound Plates The experiment deck has positions for two compound plates PLATE 1 located between the pipettor wash station and cells suspension reservoir and PLATE 2 at the right hand end of the deck 19 User s Guide for lonWorks Quattro 20 Important Ensure that compound plates are securely placed in position in the experiment deck so that the metal clamping clips are visible If not properly positioned the pipettor needles may be damaged by hitting the bottom or sides of the wells 96 and 384 well compound plates can be used in both positions Important The plate manufacturer and type must be specified in the software prior to instrument operation 2 3 7 Cell Suspension
146. ve If not you don t have the proper rights If so select Remove The uninstaller will appear Click the Next button if necessary to start the uninstaller Click Close if necessary to close the uninstaller when it is finished Install the new version Put in the CD and wait for the drive folder to open Once you see the Setup2 msi file double click on it to run the installer Select Yes to agree to the license then select the option to install for Everyone Keep clicking Next until the installer starts copying files Click the Close button when the installer is finished If the unit is not on power it up Once the unit has fully booted up and the motion stages have homed the D axis of the pipettor will go through the homing sequence start the Application The splash screen should show the new version Wait for the green connect light to come on When the Host has connected select Utilities Upgrade Firmware Wait for the dialog to appear it may take a few moments When the Firmware Upgrade Utility dialog is visible it should show the currently installed instrument Firmware version The Firmware version we are installing is shown in the dialog also labeled as Source File Version The network connection section should show that the connection to the instrument has been established Click the Terminate Firmware button to shut down the Firmware A timer will count the number of seconds It typically takes
147. w with the traces from the well and metric summary displayed A popup menu opened from the plate display has a number of useful commands Toolbuttons There are three toolbuttons in the Plate section title bar 90 System Software Toolbutton Description Switch Plate Toggles the plate view between the PatchPlate and the compound plate Gray Scale Uses gray scale rather than color coding for well identification in the plate displays Show Valid Cell Count For the compound plate only this populates each well with the number of replicates that successfully sealed i e the PatchPlate wells that received compound from the well 4 maximum that passed both the hole and seal tests Color Codes Color codes for wells in the Patch display both for the PatchPlate and compound plate indicate whether the well met or failed to meet particular criteria Color Description Dark Gray Wells that failed the hole test To view these wells select Hole Test as the Single Scan and Resistance as the metric in the Summary section Hole test criteria cannot be changed wells with resistance less than 1 MQ or greater than 10 MQ are classified as blocked Dark Blue Wells that failed the seal test To view these wells select Seal Test as the Single Scan and Resistance as the metric in the Summary section If you view pre or post compound scans you may see dark blue wells
148. with PP if the export is based on PatchPlate wells and CP if based on Compound Plate wells Browse Open the Browse dialog to select a directory in which to write the exported files 4 5 2 Configure Metric Export Dialog Configure the export of metrics data with the Configure Metric Export dialog Metrics data is exported as tab delimited text file with iha extension 101 User s Guide for lonWorks Quattro 102 You can configure automatic export of metric data within protocol definition Automatic Metric Export on the Analyze Export Data tab in the Protocol Editor dialog Or export metrics from a saved data file Export Metrics command in the File menu The same configuration dialog is used for both protocol and data file export Section Description Select Results Format PatchPlate Well Labels the exported data using PatchPlate row and column designations Compound Plate Well Labels the exported data using row and column designation from the Compound Plate Depending on the Metric Order option results from the replicate wells may be grouped together In Matrix Plate Data are organized into blocks corresponding to the dimensions of the plate format 8x48 for PatchPlate 8x12 for 96 well compound plate and 16x24 for 384 well compound plate There will be one block per scan per metric unless a command voltage family is used In Columns Data are placed vertically down the p

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User`s Guide for IonWorks® Quattro