CytoQuest CR User Manual
Contents
1. O Quick Start Custom Methiods Utilities Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 DB WAbnova www abnova com 8 1 4 Press the Custom Methods icon 8 1 5 Enter or scan in Barcode Contents ID for materials Material Parameters Barcode Contents ID Sample Capturing Antibody CytoChipNano Detecting Antibody 1 SuperBlocker scx Spiral Chamber Detecting Antibody 2 GO O Custom Methods Prime Execute Report Clean Home Exit 8 1 6 Press the Flow icon on the left side of the screen Fluidic Pump Parameters In Out A Volume uL Flow Rate mL hr A Pipette WM Pipette 2 PFA v 200 A 200 v 300 A 300 Replace NO O O Custom Methods Prime Execute Report Clean Home Exit Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 DE WAbnova KA 8 1 7 Setup the type of the fixation buffer in the Fluidic Pump Parameters Fluidic Pump Parameters In Out A Volume uL Flow Rate mL hr A Pipette WM Pipette 2 PFA pa Pipette WM Pipette Absolute Ethanol Replace Chip Remove Chip Close Receptor Separate Chamber Load Chamber2. ICON Real Time Profile Insert the SCx Spiral Chamber end tubing into the sample vial Terminate d 00H00 Custom Methods Prime Execute Report Clean Home Exit Place the SCx Spiral Chamber and the sample vial on Spiral Chamber Holder as shown below Adjust height of stand via the A N adjustment screw indicated in the red circle in order to ensure the end of tubing reach the bottom of the sample vial Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 34 Abnova MA 8 4 5 8 4 6 8 4 7 8 4 8 8 4 9 8 4 10 8 4 11 8 4 12 8 4 13 8 4 14 8 4 15 The Syringe Pump withdraws 100 uL of sample 12 0 mL hour The Syringe Pump discharges 100 uL of Wash Medium into the waste bottle via Waste Line 100 0 mL hour Follow the icon instruction in popup menu Assemble the SCx Spiral Chamber and connect to the Chamber Port gently and press the OK icon Real Time Profile 0 Custom Methods O e JA Execute Report Exit The Syringe Pump withdraws 300 uL of Wash Medium from the Wash Medium Bottle 300 0 mL hour The Microvibrator in the SCx Spiral Chamber will turn on automatically The Syringe Pump injects 300 uL of Wash Medium into the SCx Spiral Chamber via Sample Line 2 4 mL hour The Syringe Pum
3. WAbnova USER MANUAL CytoGuest CR Non invasive Circulating Rare Cell Isolation and Retrieval System mm aa wo enougy amp o pi 0 O c Hi o a www abnova com WAbnova Content Chapter 1 Safety Information Chapter 2 System Overview 2 1 Background 2 2 Description 2 3 Consumable Replacement Schedule 2 4 Workflow Chapter3 Required Materials Chapter 4 Reagent Preparation Chapter 5 Blood Collection and Handling 5 1 Phlebotomy 5 2 Transportation Chapter 6 Cell Enrichment 6 1 Leucosep Preparation Chapter 7 Capture Antibody Immobilization Chapter 8 Cell Capture 8 1 Parameters and Settings 8 2 System Priming 8 3 Buffer Filling 8 4 Sample Loading and Capture 8 5 Cell Fixation 8 6 Tubing Cleaning Chapter 9 Cell Immunostaining Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 Tel 886 2 87511888 Fax 886 2 6602 1218 www abnova com 16 17 19 21 21 21 22 22 24 25 25 28 29 33 36 37 38 29 May 2015 WAbnova MATT Chapter 10 Cell FISH 40 Chapter 11 System Maintenance 43 11 1General Maintenance 43 11 2System Cleaning 44 Chapter 12 System Specifications 45 Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6
4. spiral chamber is equipped with a non sticky coil and a self contained Microvibrator for unimpeded delivery of pretreated blood sample into the nanoarray HBx micromixer provides a herringbone conduit for cell mixing CytoChipNano is a streptavidin nanoarray which captures the CRCs for cell enumeration and single cell isolation by laser microdissection or micromanipulation CytoChipNano CR is a specialized streptavidin nanoarray with thermo sensitive coating which captures and releases the CRCs via a TCx thermal control of alternating temperatures Monoclonal antibodies targeting specific surface biomarkers of circulating rare cells can be flexibly interchanged to accommodate the biomarker switch in CRC progression This results in highly pure and viable CRCs for downstream protein characterizations gene analyses and cell assays Cell enumeration is currently the only FDA approved application as a prognostic marker in breast prostate and colorectal cancers In contrast effective single cell isolation and retrieval of CRCs opens up new scope of applications in the diagnostic and pharmaceutical industry Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 mo Ae Abnova abno com 2 2 Description Wash Medium Bottle Fluidic Bottles Bottle Reservoir Fixation Bottle YS ae Vibrato
5. NO AW amp Custom Methods Prime Execute Report Clean Home Exit e For Cell Immunostaining 2 PFA is recommended and A N optimized for CytoQuest CR e For Cell Immunostaining plus Cell FISH absolute EtOH is recommended and optimized for CytoQuest CR e For Cell Immunostaining plus Single Cell Micromanipulation 2 PFA is recommended and optimized for CytoQuest CR 8 1 8 The default temperature of the TCx Thermal Control is set at 22 C Contact an Abnova Technical Representative if you would like to alter the default setting Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 OTs Abnova MAA 8 2 System Priming 8 2 1 Press the Prime icon on the bottom tool bar to open the Prime page 8 2 2 Place the Clean Chip in the Chip Receptor Close the Chip Receptor Insert the SCx Spiral Chamber into the Chamber Port Then Insert the Sample line tubing into the SCx Spiral Chamber Press Run Button 0 OO O Execute Report Clean Home 8 2 3 Press the Run icon to start the priming and wait until this process is done Custom Methods 8 2 4 Please proceed to the 8 3 1 Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6
6. and centrifuge the probe mix 10 11 Prepare the probe mix as per manufacturer s instructions 10 12 Briefly vortex and centrifuge the tube 10 13 Denature the probe at 75 C for 5 min 10 14 Briefly centrifuge the tube Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 A1 Abnova APA 10 15 Warm up the slide on a hotplate at 45 C for 5 min before applying the probe 10 16 Apply 10 uL of the probe mix to the slide and immediately apply the coverslip on the slide 10 17 Seal the coverslip with rubber cement Co denaturation 10 18 Warm the slide on a hotplate at 80 C for 2 min for denaturation of target and probe DNA 10 19 Hybridize at 37 C for 36 48 hr Washing the slide 10 20 Carefully remove the rubber cement and coverslip by securing the coverslip between your index finger and thumb 10 21 Slowly peel off the rubber cement with forceps 10 22 Carefully slip the coverslip off the slide without lifting it 10 23 Immediately immerse the slide in 2X SSC 0 396 NP 40 at 72C for 2 min 10 24 Immerse the slide in 2X SSC at RT for 5 min 10 25 Air dry the slide in the dark and keep the slide in an upright position Counter Stain and Mounting 10 26 Apply 20 uL of DAPI counterstain to the target area and apply coverslip Place the slide at RT for 15 min 10 27 The slid
7. 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 an Om Abnova MA 7 Biological samples such as tissues body fluids and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective eyewear clothing and gloves 8 Do not attempt to reuse the consumable parts and materials Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 3 WAbnova MAA System Overview 2 1 Background Abnova s CytoQuest CR is a non invasive system for capture enumeration isolation and retrieval of circulating rare cells CRCs Three major subtypes of CRCs in translational research and clinical studies are circulating tumor cells CTCs circulating stem cells CSCs and circulating fetal cells CFCs A challenge for market adoption of CRCs is the efficient and reproducible identification single cell isolation and retrieval of highly pure and viable CRCs with their applications supported by a wide repertoire of standardized GMP grade bioreagents CytoQuest CR technology utilizes SCx spiral chamber HBx micromixer antibody immobilized nanoarray and TCx thermal control to enable a multitude of CRC functions SCx
8. 25 minutes if absolute EtOH was used as the fixation buffer Cell Capture process is now finished Please proceed to Cell Immunostaining Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 36 Abnova sain 8 6 Tubing Cleaning 8 6 1 The Syringe Pump withdraws 500 uL of deionized water 300 0 mL hour 8 6 2 The Syringe Pump discharges 500 uL of deionized water into the SCx Spiral Chamber via Sample Line 100 0 mL hour 8 6 3 The Syringe Pump withdraws 500 uL of 70 EtOH 300 0 mL hour 8 6 4 The Syringe Pump discharges 500 uL of 70 EtOH into the SCx Spiral Chamber via Sample Line 100 0 mL hour 8 6 5 The Syringe Pump withdraws 500 uL of PBS 300 0 mL hour 8 6 6 The Syringe Pump discharges 500 uL of PBS into the SCx Spiral Chamber via Sample Line 100 0 mL hour gt Please click Cell Capture for Abvideo instruction of operation Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 soJn WAbnova MAA Cell Immunostaining 9 1 Pipette 200 uL of 1X PBS into the CytoChipNano three times to remove the residual fixation buffer 9 2 Pipette 200 uL of 1X ADB 0 2 Triton X 100 into the CytoCh
9. 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 28 WAbnova 8 3 Buffer Filling 8 3 1 Press the Execute icon on the bottom tool Execute page Real Time Profile i i i i i i 0 1 200 400 600 800 1000 1200 1400 1600 1882 Time sec Remaining Time ER min Custom Methods Prime Execute Report Clean www abnova com bar to open the Terminate a AM Home Exit 8 3 2 Press the Run icon to start the capture process 8 3 3 Follow the instruction in popup menu Pipette 1 mL of Wash Medium into the Wash Medium Bottle and press the OK icon Real Time Profile Pipet 1mL of Wash Medium into the Wash Medium Bottle Remaining Time EM min o Terminate O 3 O Custom Methods Prime Execute Report lean Home Exit Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 I9 Abnova SAFE 8 3 4 Please follow the instruction in popup menu Ex Pipette 1 mL of 2 PFA into the Fixation Bottle and press the OK icon The instruction varies with the fixation buffer setting in 8 1 7 Real Time Profile Pipet 1mL of 2 PFA into the Fixation Bottle 50090900 Custom Methods Execute Report 8 3 5 The Syringe Pump withdraws 200 uL of Wash Medium 300 0 mL hour fr
10. 6 2 6602 1218 29 May 2015 16 WAbnova MAP Required Materials Catalog Brand CytoQuest Epithelial Cell Kit e Anti EpCAM capturing antibody Biotin e Anti PanCK detecting antibody FITC e Anti CD45 detecting antibody PE e 50X Antibody Dilution Buffer 50X ADB CytoQuest Epithelial Cell Kit is used for Cell Capture and Cell immunostaining described in this manual User may select other kits for different types of CRCs Please visit CytoQuest CR for more information Reagent Catalog Brand Bovine Serum Albumin BSA BP9706 100 Certified Fetal Bovine Serum FBS CytoQuest Epithelial Cell Solution e 50X Antibody Dilution Buffer 50X ADB Abnova CytoQuest SuperBlocker DAPI For Cell Immunostaining DAPI Counterstain 150ng ml For Cell FISH FFPE FISH PreTreatment Kit 1 Chemical Catalog Brand 20 Paraformaldehyde formaldehyde Aqueous pama Electron Solution Microscopy Science Dimethyl Sulfoxide DMSO IGEPAL CA 630 NP 40 Phosphate Buffered Saline PBS Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 slg es Abnova Propylene Glycol RPMI Medium Triton X 100 Consumable 15 mL Centrifuge Tubes 5 mL Dropper 50 mL Centrifuge Tubes BD Vacutainer Plastic Blood Collection Tubes with Sodium Heparin BD
11. 602 1218 E WAbnova MAA Safety Information Users should always be aware of the following general safety precautions when operating this instrument Using this instrument in a manner not specified in this manual may result in personal injury or damage to the instrument Abnova Taiwan Corp assumes no liability for the customer s failure to comply with these precautions Safety Notices 1 To prevent electric shock users should keep hands equipment clothing work areas dry at all times 2 The instrument is to be moved and positioned only by the personnel or vendor specified in the applicable site preparation guide If you decide to lift or move the instrument after it has been installed do not attempt to lift or move the instrument without the assistance of others the use of appropriate moving equipment and proper lifting techniques 3 The instrument should only be installed in the environment with good ventilation 4 The system must be installed and maintained by an Abnova Taiwan Corp Technical Representative 5 Wear appropriate personal protective equipment during operation for example safety goggles gloves or protective clothing 6 Ensure that anyone who operates the instrument has received instructions in both general safety training for laboratories and specific safety training for the instrument Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei
12. 888 Fax 886 2 6602 1218 46 Abnova Taiwan Corporation 9F No 108 Jhouzih St Neihu Taipei 114 Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218
13. Dilute 20 paraformaldehyde aqueous solution with 1X PBS 2 PFA should be aliquoted and frozen at 20 C 2 PFA can be stored at 4 C for 1 week e FM Freezing Medium FM consists of 90 FBS 5 DMSO and 5 propylene glycol FM should be aliquoted and frozen at 20 C Once thawed it can be stored at 4 C for up to 3 weeks Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 19 Abnova sain e 2X SSC Wash Buffer Dissolve 17 53 g of sodium chloride and 8 82 g of sodium citrate tribasic dehydrate in 700 mL deionized water Adjust the pH to 7 0 and the volume to 1 L Following autoclaving sterilization 2X SSC Wash Buffer can be stored at ambient temperature e 2X SSC 0 3 NP 40 Add 3mL of IGEPAL CA 630 to 990mL of 2X SSC Wash Buffer pre warmed to 70 C Bring the final volume to 1 L with 2X SSC Wash Buffer Store 2X SSC 0 3 NP 40 at ambient temperature Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 Tel 886 2 87511888 Fax 886 2 6602 1218 29 May 2015 20 WAbnova www abnova com Blood Collection and Handling 5 1 Phlebotomy Access to a peripheral vein should be performed with a 23G needle The first 2 mL of collected blood should be discarded to avoid potent
14. L tube 6 1 9 Wash the PBMCs with 10 mL of WM 6 1 10 Centrifuge at 300 x g and 4 C for 10 min then aspirate and discard the supernatant 6 1 11 Re suspend the cell pellet with 1 mL WM and transfer the cell suspension to microcentrifuge tube 6 1 12 Centrifuge microcentrifuge tube at 300 x g and 4 C for 10 min then aspirate and discard the supernatant 6 1 13 For cell enumeration re suspend the cell pellet with 105 uL of WM and proceed to the protocol of Cell Capture Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 m DO m Abnova www abnova com rug Please click Leucosep Preparation for Abvideo instruction of operation Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 93 WAbnova KAPEWA Capture Antibody Immobilization 7 1 Wash the CytoChipNano with 200 uL of ethanol followed by 200 uL of 1X PBS twice Make sure no bubble exists in the microfluidic channel 7 2 Prepare Capture Antibody Working Solution by adding 50 uL of Anti EpCAM capturing antibody Biotin to 50 uL of 1X PBS 7 3 Pipette 100 uL of Capture Antibody Working Solution into the chip and incubate the chip at RT for 1 hour in humidity bo
15. Vacutainer Safety Lok Blood Collection Sets with Pre Attached Holder Bottle and Tubing Pack Coverslip Cryovial CytoChipNano CytoChipNano Combo Pack e CytoChipNano e SCx Spiral Chamber e Plugs Eppendorf Micro Test Tubes Mr Frosty Freezing Container www abnova com 93426 100ML Sigma Aldrich Catalog Brand 30006 3381 Alpha Plus vee eee aaa E 0030 121 589 Eppendorf 15 350 50 Fishersci Histopaque 1077 10771 Sigma Aldrich Rubber Cement CXA 100 Kokuyo SCx Spiral Chamber 00314 Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan Tel 886 2 87511888 Fax 886 2 6602 1218 PP PLM A 027 Version 1 6 29 May 2015 18 WAbnova KA Reagent Preparation N All the reagents solutions used should be of molecular grade and sterile filtered e 1X PBS 1X PBS consists of 8 1 mM NasHPO 2H20 1 46 mM KH2PO 138 mM NaCl 2 7 mM KCI pH 7 4 at 25 C 1X PBS without calcium and magnesium can be stored at room temperature e WM Wash Medium RPMI with 5 FBS WM is aliquoted and kept frozen at 20 C Once thawed WM can be stored at 4 C for up to 3 weeks e 1X ADB Dilute 50X ADB by adding adequate 1X PBS to 50X ADB 1X ADB should be kept on ice or at 4 C and used within 2 days e 1X ADB 0 2 Triton X 100 Add 0 2 mL of Triton X 100 to 100 mL of 1X ADB e 2 PFA 2 Paraformaldehyde Aqueous Solution
16. bar to open the Clean Page 11 2 2 Place the Clean Chip into the Chip Receptor Press Run Button Terminate Remaining Time Ea min Custom Methods Prime Execute Report Clean Home Exit 11 2 3 Press the Run icon to start the cleaning process and wait until it is done 11 2 4 Turn off the power switches of the liquid handler and the thermal control of CytoQuest CR system i Please click System Maintenance for Abvideo instruction of operation Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 44 WAbnova sn sbnova com System Specifications Hardware Specifications General Specifications Dimensions 565 06mm h x 340 8mm d x 271 8mm w Liquid Handler Unit 100 240VAC 50 60Hz 1 6A Thermo Control Unit 85 250VAC 50 60Hz 4 0A Liquid Handler Unit 1 6A Peak 250V Replace Fuse Thermo Control Unit 4A Peak 250V Operating Temperature 60 F to 104 F 15 C to 40 C Programable Thermo 4 40 C Variable Time Span Control Profile Environmental Specifications Storage Temperature 5 F to 158 F 20 C to 70 C Operation Altitude Up to 2000m Operation Environment For Indoor Use Operating Humidity 20 to 90 Relative Humidity Non Condensing Storage Humidity 20 to 95 Relative Humidity Non Condensing Copyright A
17. bnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 45 Abnova www abnova com Software Specifications General Specifications Scanner Function Status Display System Clean Report Generation Necessary Driver Scanning Capability to Read QR Code Barcode Progress and Fluid Handling Prime and Clean Print and Export File LabVIEW Run Time Engine 2011 SP1 Standard RTE NI VISA Run Time Engine 5 4 ATEN 32 64bit Driver v2 0 7 0 Binary Touchscreen Laptop Specifications General Specifications Operating System Processor Memory Free Disk Space Interfaces USB Ports Symbols tial Date of manufacture Protective Conductor Manufacturer Windows 8 7 Vista 32 bit and 64 bit Windows XP SP3 32 bit Windows Server 2003 R2 32 bit Windows Server 2008 R2 64 bit Pentium Ill Celeron 866 MHz or Equivalent 256MB Minimum 512MB Recommended 1GB USB 3 0 Port 1 Serial Electronic sn Number ui specification Consult A Caution pR instructions for use X WEEE ji ji Abnova Taiwan Corporation 3rd FI No 132 Xiyuan Rd Zhongli District Taoyuan City Taiwan Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511
18. e is now ready for examination under fluorescence microscope gs Please click Cell FISH for Abvideo instruction of operation Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 ADA WAbnova MA System Maintenance 11 1 General Maintenance For reliable operation keep the instrument free from dust and liquid spills We recommend cleaning the instrument periodically A soft cloth dampened in mild detergent is sufficient Please operate the instrument at least once a year If any surfaces have been contaminated with bio hazardous material a sterilizing solution must be used Buffer bottles on top of the instrument are filled with DI water yellow bottle tubing 70 ethanol green bottle tubing and 1X PBS blue bottle tubing The white instrument tubing is washed by these buffers To avoid contamination wash buffer bottles and replace the content with fresh buffers once a week For conduct maintenance users should always perform System Cleaning after operation Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 43 Abnova Susanna 11 2 System Cleaning 11 2 1 Press the Clean icon on the bottom tool
19. euse Every use Chip Opener Razor Reusable Replace if malfunction These one time use consumables come together with CytoChipNano Combo Pack CAT KA4440 Abnova Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan Tel 886 2 87511888 Fax 886 2 6602 1218 PP PLM A 027 8 Version 1 6 29 May 2015 Y 9 Abn ova www abnova Com 2 3 1 Removing Connecting Base from SCx Spiral Chamber Top to bottom Hold the Microvibrator and pull off the Connecting Base Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 Q Abnova su sbnova com 2 3 2 Removing Electrorod from Connecting Base Top to bottom Use finger to remove Electrorod from used Connecting Base Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 10 WAb n ova www abnova com 2 3 3 Removing Inlet Plug and Outlet Plug from the Coupler Insert the tip of the tweezers into the end of plugs at the underside of the Coupler Loosen the plugs by gently twisting and shaking Then pull the plugs out with one hand while supporting the Coupler with the opposite
20. hand Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Tel 886 2 87511888 Fax 886 2 6602 1218 Version 1 6 29 May 2015 411 WAbnova aes 2 3 4 Inserting Inlet Plug and Outlet Plug into the Coupler Outlet Plug White Inlet Plug Blue Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 12 Abnova sain Top to bottom 1 Open the Coupler and prepare Inlet Plug blue and Outlet Plug white by cleansing and lubricating the plugs with 70 ethanol first 2 Align Inlet Plug with the blue circled hole and Outlet Plug with the red circled hole as shown and then fully insert the plugs from the underside of the Coupler 3 Close the Coupler gently Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 wana WAb n ova www abnova com 2 3 5 Assembling SCx Spiral Chamber Top to bottom 1 Prepare Coil Holder Microvibrator and Connecting Base of SCx Spiral Chamber Assemble firmly Microvibrator into Coil Holder Assemble firmly the part from step 2 into Connecting Base while pass the tubing through the cente
21. ial skin cell contamination from venipuncture Then transfer 10 mL of blood sample in each Heparin tube green cap Gently rotate the 10 mL Heparin tube end over end five times immediately Place the collected blood sample at room temperature before use 5 2 Transportation Blood has a maximum preservation time of 6 hours at room temperature For example if the blood is drawn at 9am in a hospital it should be processed by 3 pm Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 a94 ss WAbnova MA Cell Enrichment 6 1 Leucosep Preparation 6 1 1 Warm up Histopaque 1077 density gradient media to room temperature RT 6 1 2 Fill each Leucosep tube with 3 mL of Histopaque 1077 density gradient media 6 1 3 Centrifuge the Leucosep tube at 1000 x g and RT for 30 seconds The density gradient media should be spun below the porous barrier 6 1 4 Dilute the blood sample at 1 1 ratio with 1X PBS 6 1 5 Carefully pour 4 mL of diluted blood sample into the prepared Leucosep tube 6 1 6 Centrifuge the Leucosep tube at 1000 x g and RT for 10 minutes in a swinging bucket rotor 6 1 7 Aspirate and discard the top layer plasma solution to a remnant of 5 to 10 mm in height above the PBMCs interphase 6 1 8 Carefully transfer the PBMCs layer above the porous barrier to a new 15 m
22. ipNano for 20 minutes 9 3 Pipette 200 uL of 1X PBS into the CytoChipNano three times to remove 1X ADB 0 2 Triton X 100 9 4 Prepare the detecting antibody cocktail by adding 6 25 uL of Anti PanCK detecting antibody FITC and adding 1 25 uL of Anti CD45 detecting antibody PE to 250 uL of 1X ADB 9 5 Pipette 250 UL of detecting antibody cocktail into the CytoChipNano and incubate it away from light at room temperature for 1 hour in humidity box 9 6 Pipette 200 uL of 1X PBS into the CytoChipNano three times to remove the residual detecting antibody cocktail 9 7 Pipette 200 uL of 300 nM DAPI solution into the CytoChipNano and wait for 10 minutes 9 8 Pipette 200 uL of 1X PBS into the CytoChipNano to remove the residual DAPI solution and incubate it away from light at room temperature 9 9 Cell Immunostaining in the CytoChipNano is now ready for Cell FISH or microscope observation Please refer to recommended filter sets listed for each fluorochrome below For single cell micromanipulation please refer to CytoQuest Single Cell Picking Kit http www abnova com protocol pdf KA4578 pdf Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 38 WAbnova aan Fluorochrome EK nm EM nm Recommended Filter Set APC Cy5 4040C SemRock 3 4 DAPI 49000 Chroma R phycoeryth
23. low the instruction in popup menu Close the Chip Receptor and press the OK icon Real Time Profile Remaining Time O min NG Ce s dA Custom Methods Execute Report Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 39 WAbnova 8 4 Sample Loading and Capture 8 4 1 8 4 2 8 4 3 Follow the icon instruction in popup menu Disconnect the SCx Spiral Chamber from the Chamber Port Separate the Connecting Base hold the Coil Holder and press the OK icon Do not disconnect the SCx Spiral Chamber and Sample Line Do not insert the SCx Spiral Chamber end tubing into the sample vial yet Real Time Profile O e WA Execute Report Sle Exit O The Syringe Pump withdraws 20 uL of Wash Medium from Wash Medium Bottle 300 0 mL hour The Syringe Pump injects 20 uL of Wash Medium into the SCx Spiral Chamber via Sample Line 12 0 mL hour Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 33 www abnova com WAbnova su sbnova com 8 4 4 Follow the instruction in popup menu Insert the SCx Spiral Chamber end tubing into the sample vial and press the OK
24. om the Wash Medium Bottle 8 3 6 The Syringe Pump discharges 200 uL of Wash Medium into the waste bottle via Waste Line 100 0 mL hour 8 3 7 The Syringe Pump withdraws 300 uL of Wash Medium 300 0 mL hour from the Wash Medium Bottle 8 3 8 The Syringe Pump injects 300 uL of Wash Medium into the SCx Spiral Chamber via Sample Line 100 0 mL hour Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 30 WAbnova BANGIN 8 3 9 Follow the icon instruction in popup menu Open the Chip Receptor Remove the Clean Chip and place capture antibody coated CytoChipNano Press the OK icon e Donot close the Chip Receptor e Place and push the CytoChipNano forward until it touches the opposing edge of the Chip Receptor Real Time Profile Remaining Time EG min Custom Methods Prime Execute Report Clean Home Exit Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 31 Abnova MA 8 3 10 The Syringe Pump withdraws 10 uL of Wash Medium 300 0 mL hour from the Wash Medium Bottle 8 3 11 The Syringe Pump injects 10 uL of Wash Medium into the SCx Spiral Chamber via Sample Line 12 0 mL hour 8 3 12 Fol
25. p withdraws 960 uL of PBS 300 0 mL hour The Syringe Pump injects 960 uL of PBS into the SCx Spiral Chamber via Sample Line 4 8 mL hour The Syringe Pump withdraws 300 uL of PBS 300 0 mL hour The Syringe Pump injects 300 uL of PBS into the SCx Spiral Chamber via Sample Line 6 0 mL hour The Microvibrator in the SCx Spiral Chamber will turn off automatically Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 35 Abnova sain 8 5 Cell Fixation 8 5 1 8 5 2 8 5 3 8 5 4 8 5 5 8 5 6 8 5 7 The Syringe Pump withdraws 200 yL of the fixation buffer from the Fixation Bottle 300 0 mL hour The Syringe Pump discharges 200 uL of the fixation buffer into the waste bottle via Waste Line 100 0 mL hour The Syringe Pump withdraws 400 uL of the fixation buffer from the Fixation Bottle 800 0 mL hour The Syringe Pump injects 400 uL of the fixation buffer into the SCx Spiral Chamber via Sample Line 2 PFA 4 8 mL hour absolute EtOH 2 4 mL hour Follow the icon instruction in popup menu Replace the CytoChipNano with Clean Chip and press the OK icon Real Time Profile Custom Methods Prime Execu Report Clean Allow the CytoChipNano to sit for 15 minutes if 2 PFA was used as the fixation buffer Allow the CytoChipNano to sit for
26. r Voltage Fluidic Pump Display USB Port RS232 Port Syringe Pump SCx Spiral Chamber iN Temperature Coupler Spay Thermal Chip Receptor Controller Liquid Handler Power Switch MAN Va Power Switch TCx Thermal Chuck lt YN a N AC Power Barcode Scanner Socket Waste Bottle Vial Holder CytoQuest CR system SCx spiral chamber HBx micromixer and TCx thermal control are trademarks and pending patents of Abnova Taiwan Corporation Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 Ha Abn ova www abnova com DI Water Yellow PBS 70 EtOH Blue Green Fixation Wash Medium Red Bottle View Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 6 WAbnova www abnova com PBS Line R Blue DI Water Line Yellow Wash Medium Line 70 EtOH Line Red Green Fixation Line Air Sample Line Waste Line Fluidic view Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 n
27. r of Connecting Base Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 14 Abn ova www abnova com 2 3 6 Inserting SCx Spiral Chamber into the Chamber Port of the Coupler Left to right Prepare SCx Spiral Chamber Connect Sample Line to SCx Spiral Chamber Insert SCx Spiral Chamber into the Chamber Port of nA 2 NN the Coupler Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 15 2 Abnov n ova www abnova com 2 4 Workflow Chapter 5 Blood Collection Chapter 6 1 Chapter 7 Capture Antibody Immobilization Operating time 60 min Leucosep Preparation Operating time 30 min Chapter 8 CytoQuest CR Cell Capture Operating time 45 min chip v Cell Immunostaining Operating time 90 min Chapter 10 l Cell FISH Operating time 50 hr Chapter 9 v Microscope Examination Operate in a clean laboratory environment E Operate inside CytoQuest CR Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 Tel 886 2 87511888 Fax 88
28. rin PE 49010 Chroma 650 660 59 461 565 578 4 ss Please click Cell Immunostaining for Abvideo instruction of operation Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 39 Abnova abno com Cell FISH Slide preparation 10 1 Prepare 200 uL of air into the CytoChipNano twice to remove 1X PBS 10 2 Flip the CytoChipNano so the bottom side faces up There are 2 semi circular notches Gently insert the tip of the tweezer into the notch then lift the CytoChipNano up from the Bento Cover below 10 4 Place the CytoChipNano right side up on the indent Hold down the ends of the CytoChipNano and cut the four sides of the CytoChipNano with sliding blade Do not press the middle of the CytoChipNano Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 AQ Abnova eae Slide pre treatment 10 6 10 7 10 8 10 9 Immerse the slide in 2X SSC at room temperature for 15 min Immerse the slide in 2X SSC at 37 C for 30 min Dehydrate the slide by immersing the slide in a series of EtOH solutions 70 85 and 100 2min each at RT Air dry the slide Probe preparation 10 10 Briefly vortex
29. un Ta WAbnova 2 3 Consumable Replacement Schedule www abnova com To avoid cross contamination user should replace CytoChipNano Coil Holder Connecting base of SCx Spiral Chamber Intlet Plug and Outlet Plug when applying new sample in Cell Capture See Consumable Replacement Schedule below Sub Items Replacement Schedule GAIN 70 EtOH Bottle Reusable System notice DI Water Bottle Reusable System notice Fluidic Bottles PBS Bottle Reusable System notice Wash Medium Bottle Reusable System notice Fixation Bottle Reusable System notice 70 EtOH Line Green Reusable System notice DI Water Line Yellow Reusable System notice KA4442 PBS Line Bue Reusable System notice Fluidic Tubings Wash Medium Line Red Reusable System notice Fixation Line Reusable System notice Sample Line Reusable System notice Waste Line Reusable System notice Air filter Reusable System notice CytoChipNano Do not reuse Every use U0095 Chips CytoChipNano CR Do not reuse Every use Clean Chip Reusable Every 20 uses Dummy Chip Reusable Every 20 uses Coil Holder Do not reuse Every use TV SCx Spiral Connecting Base Do not reuse Every use Chamber Microvibrator Reusable Replace if malfunction Electrorod Reusable Replace if malfunction Inlet Plug Blue Do not reuse Every use Plugs Outlet Plug White Do not reuse Every use KA4441 a i Blue with Do not r
30. x Make sure the inlet and outlet of the chip are consistently covered with working solution throughout incubation 7 4 Wash the CytoChipNano with 200 uL of 1X PBS three times to remove the residual Capture Antibody Working Solution 7 5 Prepare CytoQuest SuperBlocker Working Solution by adding 10 uL of SuperBlocker to 90 uL of 1X PBS 7 6 Pipette 100 uL of CytoQuest SuperBlocker Working Solution into the CytoChipNano and incubate the CytoChipNano at RT for 1 hour in humidity box Make sure the inlet and outlet of the chip are consistently covered with working solution during incubation 7 7 Wash the CytoChipNano with 200 uL of 1X PBS three times to remove the residual CytoQuest Super Blocker Working solution 7 8 The CytoChipNano is now coated with capture antibody and is ready for use wag Pee click Capture Antibody Immobilization for Abvideo instruction of operation Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 6 29 May 2015 Tel 886 2 87511888 Fax 886 2 6602 1218 94 WAbnova www abnova com Cell Capture 8 1 Parameters and Settings 8 1 1 Turn on the power switches of the liquid handler and thermal control of CytoQuest CR system 8 1 2 Turn on the computer and press the S13001SP01 CytoQuest CR v2 9 icon Q Abnova Enter Operator ID Exit 8 1 3 Enter or scan in Operator ID
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