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CytoQuest CR Manual

1. www abnova com 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com DI Water Yellow PBS 70 EtOH Blue Green Wash Medium Red Buffer Bottle Copyright Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 5 www abnova com 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com ike 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 OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 6 www abnova com 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com Assembling SCx Spiral Chamber top to bottom 1 Prepare Coil Holder Micro vibrator and Connecting Base of SCx Spiral Chamber Assemble Micro vibrator into Coil Holder Assemble the part from step 2 into Connecting Base Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM
2. 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 OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 3 Abnova conan com 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com Description Wash Medium Bottle Buffer Bottle Bottle Reservoir Fixation Bottle Vibrator Voltage Display mango wame SSB po RS232 Port SCx Spiral Chamber Temperature Coupler Display Liquid Handler Chip Receptor Power Switch TCx Thermal Control A Ng Barcode Scanner AC Power Socket Thermal Control Power Switch aste Vial Holder Bottle Front view Back view 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 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 sAm
3. 10 22 The slides are now ready for examination under fluorescence microscope Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan Tel 886 2 87511888 Fax 886 2 6602 1218 PP PLM A 027 Version 1 3 28 Nov 2014 33 Abnova a 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com 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 OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 34 Abnova conan com 9F
4. No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com 11 2 System Cleaning 11 2 1 Press the Clean icon on the bottom tool bar to open the Clean Page 11 2 2 Place the Clean Chip into the Chip Receptor gt 4 Press Run Button Remaining Time min Custom Methods Prime Execute Report Clean 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 thermal control of CytoQuest CR system Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 35 Abnova www abnova com 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com System Specifications Hardware Specifications Operating 60 F to 95 F 15 C to 35 C Temperature Programable Thermo 4 40 C Variable Time Span Control Profile Storage Temperature Operation Altitude Up to 2000m Operation 5 F to 158 F 20 C to 70 C For Indoor Use Environment Operating Humidity 20 to 90 Relative Humidity Non Condensing Storage Humidity 20 to 95 Relative Humidity Non Condensing Copyright Abnova Taiwan Corporation Authorized use only OF
5. Real Time Profile Pipette 1mL of Absolute Ethanol into the Fixation Bottle min us s Prime Execute Report Clean Exit 8 3 5 The Syringe Pump withdraws 200 uL of Wash Medium 300 0 mL hour from 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 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 24 WAbnova www abnova com 9F No 108 Jhouzih St Taipei Taiwan Tel 4 886 2 8751 1888 Fax 4 886 2 6602 1218 E mail sales abnova com 8 3 9 Follow the instruction in popup menu Open the Chip Receptor Remove the Clean Chip and replace with capture antibody coated CytoChipNano and press the OK icon Real Time Profile e N a 20 Temperature C a oO O wm Terminate Remaining Time Ea min Custom Methods Report Clean Exit 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 m
6. Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 Pome ee WAbnova www abnova com 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com 10 4 Immerse the slide in 2X SSC Wash Buffer at RT for 15 minutes 10 5 Immerse the slide in 2X SSC Wash Buffer at 37 C for 30 minutes 10 6 Immerse the slide in 70 Ethanol at RT for 2 minutes 10 7 Immerse the slide in 85 Ethanol at RT for 2 minutes 10 8 Immerse the slide in 100 Ethanol at RT for 2 minutes 10 9 Leave the slide air dried 10 10 Denature the FISH probe by incubating it in water bath at 75 C for 5 minutes 10 11 Before applying the FISH probe warm up the slide on the hot plate at 45 C for 5 minutes 10 12 Immerse the target area with 10ul of denatured FISH probe and cover it with 22 mm x 22 mm coverslip 10 13 Seal the coverslip with rubber cement 10 14 Co denature the slide on the hot plate at 80 C for 2 minutes 10 15 Incubate the slide in the humidity chamber pre warmed at 37 C for 16 48 hours 10 16 Gently remove the rubber cement and coverslip 10 17 Immerse the slide in 2X SSC 0 3 NP 40 at 72 C for 2 minutes 10 18 Immerse the slide in 2X SSC at RT for 5 minutes 10 19 Leave the slide air dried in the dark 10 20 Apply 10uL of DAPI 150ng ml solution to the target area and cover it with coverslip 10 21 Leave the slide at 20 C for more than 30 minutes
7. 08 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 D WAbnova www abnova com 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com 8 3 Filling Buffer 8 3 1 Press the Execute icon on the bottom tool bar to open the Execute page Real Time Profile 8 25 2 o w e 8 15 v p ad Oo co o 2000 4000 6000 8000 Time sec terminate temaining Tim min Custom Methods Prime Execute Report Clean 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 Pipette 1mL of Wash Medium into the Wash Medium Bottle Remaining Time min WA Custom Methods Prime Execute Report Clean Exit Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 23 Abnova semana com 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com 8 3 4 Follow the instruction in popup menu Pipette 1 mL of absolute EtOH into the Fixation Bottle and press the OK icon
8. 19 WAbnova www abnova com 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com 8 1 4 Press the Custom Methods icon Material Parameters Antibody Kit Solution Kit Capture Antibody SCx Spiral Chamber ID 10X RBC Lysis Buffer oO amp Custom Methods Prime Execute Report Clean Exit 8 1 5 Enter or scan in Sample ID CytoChipNano ID SCx Spiral Chamber ID Antibody Kit ID and Solution Kit ID 8 1 6 Press the Flow icon on the left side of the screen Fluidic Pump Parameters 3 In Out V A Volume uL Flow Rate mL hr A Pipette WM Main Pipette Absolute Ethanol 200 gt Flow 200 300 300 Temp Replace O G WAN WA Custom Methods Prime Execute Report Clean Exit Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 0 Abnova a 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com 8 1 7 Setup the Fluidic Pump Parameters Fluidic Pump Parameters In Out V A Volume uL Flow Rate mL hr A Pipette WM Pipette Absolute v ute Ethanol nang Pipette WM Pipette 4 PFA Replace Chip Remove Chip M Close Receptor Separate Chamber Load Chamber
9. 2 1218 E mail sales abnova 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 Icon Real Time Profile Saeg Insert the SCx Spiral Chamber end tubing into the sample vial K 2 kui 2 1 D a Oo o wo Custom Methods Prime Report Clean 8 4 5 The Syringe Pump withdraws 100 uL of sample 12 0 mL hour 8 4 6 The Syringe Pump discharges 100 uL of Wash Medium into the waste bottle via Waste Line 100 0 mL hour 8 4 7 Follow the instruction in popup menu Assemble the SCx Spiral Chamber and connect to the Chamber Port and press the OK icon Real Time Profile Remaining Time o fia OR TORORO Custom Methods Prime Execute Report Clean Exit Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 Abnova meman 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com 8 4 8 The Syringe Pump withdraws 300 uL of Wash Medium from the Wash Medium Bottle 300 0 mL hour 8 4 9 The Micro vibrator in the SCx Spiral Chamber will turn on automatically 8 4 10 The Syringe Pump injects 300 uL of Wash Medium into the SCx Spiral Chamber via Sample Line 2 4 mL hour 8 4 11 Th
10. 32 System Maintenance 34 General Maintenance 34 System Cleaning 35 System Specifications 36 Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 7 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 Abnova maaa 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com 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 approp
11. 511888 Fax 886 2 6602 1218 13 Abnova a 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com 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 S SC 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 OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 14 Abnova KAPAR 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com Blood Collection and Handling Phlebotomy Access to a peripheral vein should be performed with a 23G needle Collect 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 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 pr
12. A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 ST www abnova com 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com Disassembling SCx Spiral Chamber top to bottom 1 Hold the Micro vibrator and pull off the Connecting Base Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 8 Abnova www abnova com 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com Inserting SCx Spiral Chamber into Chamber Port of the Coupler left to right 1 Prepare SCx Spiral Chamber 2 Connect Sample Line to SCx Spiral Chamber 3 Insert SCx Spiral Chamber into the Chamber Port of the Coupler Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 9 Abnova www abnova com 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com Workflow Blood Collection CytoQuest CR CTC Capture Operating time 30 min chip O OTEO D Operate in a clean laboratory env
13. Abnova Taiwan Corporation Authorized use only 9F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 29 Abnova a 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com 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 Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 30 Abnova maaa 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com CTC 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 CytoChipNano for 20 mi
14. F No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 11 Abnova maman 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com e 2 Propanol CAT 109827 1L Fishersci e Bovine Serum Albumin BSA CAT BP9706 100 Fishersci e 20 Paraformaldehyde formaldehyde Aqueous Solution CAT 15715 S Electron Microscopy Science e Triton X 100 CAT 93426 100ML Sigma Aldrich e CytoQuest Epithelial Cell Kit v1 0 CAT KA4439 Abnova v Mouse Anti EpCAM capturing antibody Biotin v Mouse Anti PanCK detecting antibody FITC v Mouse Anti CD45 detecting antibody PE v 50X Antibody Dilution Buffer 50X ADB e CTC Solution Kit CAT KA3967 Abnova v 50X Antibody Dilution Buffer 50X ADB e Cartridge Pack CAT KA4440 Abnova v CytoChipNano v SCx Spiral Chamber e Certified Fetal Bovine Serum FBS CAT 14 502F Lonza e DAPI for CTC Immunostaining CAT D1306 Invitrogen e Ethanol CAT 100983 Merck e Coverslip CAT 990 Assistent e IGEPAL CA 630 CAT l 3021 Sigma Aldrich e Rubber Cement CAT CXA 100 KOKUYO e DAPI Counterstain 150ng ml for CTC FISH CAT U0030 Abnova e Precaution Y Be sure to read the entire manual before running the assay Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu Distric
15. Kepiace WAN amp amp Custom Methods Prime Execute Report Clean Exit Note J e For CTC Immunostaining alone 4 PFA fixation buffer is recommended and optimized for CytoQuest CR e For CTC Immunostaining plus CTC FISH absolute EtOH is recommended and optimized for CytoQuest CR e For other staining applications more testing and optimization would be required 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 OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 1 Abnova semana com 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com 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 Insert the SCx Spiral Chamber into the Chamber Port Then Insert the Sample line tubing into the SCx Spiral Chamber 4 Press Run Button G9 2 Custom Methods Prime Execute Report Clean Exit 8 2 3 Press the Run icon to start the priming and wait until this process is done Copyright Abnova Taiwan Corporation Authorized use only OF No 1
16. L hour 8 3 12 Follow the instruction in popup menu Close the Chip Receptor and press the OK icon Real Time Profile Temperature C an 8 o Oo a t Terminate Remaining Time o fim NEON Custom Methods Execute Report Clean Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 5 Abnova semana com 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com 8 4 Sample Loading and Capture 8 4 1 Follow the instruction in popup menu Disconnect the SCx Spial 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 Real Time Profile 8 4 2 The Syringe Pump withdraws 20 uL of Wash Medium from Wash Medium Bottle 300 0 mL hour 8 4 3 The Syringe Pump injects 20 uL of Wash Medium into the SCx Spiral Chamber via Sample Line 12 0mL hour Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 6 WAbnova www abnova com 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 660
17. No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 36 www abnova com Abnova 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com Software Specifications Scanner m Scanning Capability to Read QR Code Barcode Function Status Display Progress and Fluid Handling System Clean Prime and Clean Report Print and Export File Generation LabVIEW Run Time Engine 2011 Standard RTE Necessary Driver NI VISA Run Time Engine 5 4 PL2303 Prolific Driverlnstaller_v1 9 0 Touchscreen Laptop Specifications 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 Operating System Processor Pentium IIl Celeron 866 MHz or Equivalent Memory 256MB Minimum 512MB Recommended Free Disk Space 1GB Interfaces USB Ports USB 3 0 Port 1 Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 pi OT es
18. USER MANUAL CytoQuest CR Non invasive Circulating Rare Cell Isolation and Retrieval System 2014 Thermo Fisher Scientific Inc All rights reserved Trademarks used are owned as indicated at www fishersci com trademarks In the United States In Canada For customer service call 1 800 766 7000 For customer service call 1 800 234 7437 To fax an order use 1 800 926 1166 To fax an order use 1 800 463 2996 To order online www fishersci com To order online www fishersci ca eaouqy amp YD ISOENHORZAD A Thermo Fisher Scientific Brand Abnova Abnova Taiwan Corporation Taiwan R O C Content Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 6 1 6 2 Chapter 7 Chapter 8 8 1 8 2 8 3 84 8 5 8 6 Chapter 9 Chapter 10 Chapter 11 11 1 11 2 Chapter 12 www abnova com 9F No 108 Jhouzih St Taipei Taiwan 3rd FI Neo 132 Siyuan Rd Jhongl City Taoyuan County Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com Safety Information 1 System Overview 3 Required Materials 11 Reagent Preparation 13 Blood Collection and Handling 15 CTC Enrichment 16 Leucosep Preparation 16 Post Leucosep Freezing and Banking 17 Capture Antibody Immobilization 18 CTC Capture 19 Parameters and Settings 19 System Priming 22 Filling Buffer 23 Sample Loading and Capture 26 Cell Fixation 29 Tubing Cleaning 30 CTC Immunostaining 31 CTC FISH
19. banking please refer to Post Leucosep Freezing and Banking Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 16 Abnova meman 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com 6 2 Post Leucosep Freezing and Banking 6 2 1 Pre cool cryogenic freezing vials at 80 C prior to cell freezing and banking 6 2 2 Re suspend the PBMCs from step 5 1 11 with 1 mL of FM pre chilled at 4 C 6 2 3 Aliquot 0 5 mL of mixed cell Suspension to each cryogenic freezing vial 6 2 4 Keep cryogenic freezing vials in a freezing container and place at 80 C for at least 2 hours or overnight preferred 6 2 5 Transfer cryogenic freezing vials to liquid nitrogen storage within 72 hours Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 1 Abnova maman 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com 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 Ant
20. e Syringe Pump withdraws 960 uL of PBS 300 0 mL hour 8 4 12 The Syringe Pump injects 960 uL of PBS into the SCx Spiral Chamber via Sample Line 4 8 mL hour 8 4 13 The Micro vibrator in the SCx Spiral Chamber will turn off automatically Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 8 WAbnova www abnova com 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com 8 5 Cell Fixation 8 5 2 The Syringe Pump withdraws 200 uL of absolute EtOH from the Fixation Bottle 300 0 mL hour 8 5 3 The Syringe Pump discharges 200 uL of absolute EtOH into the waste bottle via Waste Line 100 0 mL hour 8 5 4 The Syringe Pump withdraws 400 uL of absolute EtOH from the Fixation Bottle 300 0 mL hour 8 5 5 The Syringe Pump injects 400 uL of absolute EtOH into the SCx Spiral Chamber via Sample Line 4 8 mL hour 8 5 6 Follow the instruction in popup menu Replace the CytoChipNano with Clean Chip and press the OK icon Real Time Profile e SRE Gi E mm A E mi O A oT Custom Methods 8 5 7 Allow the CytoChipNano to sit for 25 minutes to finish the fixation process 8 5 8 The capture process is now finished Please proceed to CTC Immunostaining Copyright
21. ibody Working Solution by adding 50 uL of mouse 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 Make sure the inlet and outlet of the chip are consistently covered with working solution throughout incubation 7 4 Wash the chip with 200 uL of 1X PBS three times to remove the residual Capture Antibody Working Solution 7 5 The CytoChipNano is now coated with capture antibody and is ready for use Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 18 Abnova www abnova com 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com CTC 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 CytoQuest CR_v2 4 icon Q Abnova Enter Operator ID Exit 8 1 3 Enter or scan in Operator ID O O Quick Start Custom Metfiods Utilities Exit Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218
22. ironment D 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 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 10 Abnova a 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com Required Materials e CytoQuest CR CAT M0014 02 Abnova e CytoChipNano CAT U0095 Abnova e SCx Spiral Chamber CAT U0314 Abnova e Coupler Plug CAT U0315 Abnova e BD Vacutainer Plastic Blood Collection Tubes with Sodium Heparin CAT 02 689 6 Fishersci e BD Vacutainer Safety Lok Blood Collection Sets with Pre Attached Holder CAT 02 683 21 Fishersci e Leucosep CAT 163290P Greiner Bio One e Histopaque 1077 CAT 10771 Sigma Aldrich e Phosphate Buffered Saline PBS CAT P 5368 Sigma Aldrich e RPMI Medium CAT 12633 012 Invitrogen e Dimethyl Sulfoxide DMSO CAT TS 20684 Fishersci e Propylene Glycol CAT 0215195790 Fishersci e 5 mL Dropper CAT 30006 3851 Alpha Plus e Cryovial CAT 02 912 729 Fishersci e 50 mL Centrifuge Tubes CAT 352070 Fishersci e 15 mL Centrifuge Tubes CAT 3131 345 008 Labcon e Eppendorf Micro Test Tubes CAT 0030 121 589 Eppendorf e Mr Frosty Freezing Container CAT 15 350 50 Fishersci Copyright Abnova Taiwan Corporation Authorized use only O
23. ng 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 spiral chamber is equipped with a non sticky coil and a self contained micro vibrator 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 gold 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
24. nutes 9 3 Prepare the detecting antibody cocktail by adding 6 25 uL of mouse Anti PanCK detecting antibody FITC and 1 25 uL of mouse Anti CD45 detecting antibody PE to 250 uL of 1X ADB 9 4 Pipette 250 UL of detecting antibody cocktail into the CytoChipNano and incubate it at RT for 1 hour 9 5 Pipette 200 uL of 1X PBS into the CytoChipNano three times to remove the residual detecting antibody cocktail 9 6 Pipette 200 uL of 300 nM DAPI solution into the CytoChipNano and wait for 10 minutes 9 7 Pipette 200 uL of 1X PBS into the CytoChipNano to remove the residual DPAI solution 9 8 CTC Immunostaining in the CytoChipNano is now ready for CTC FISH or microscope observation Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 31 WAbnova www abnova com 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com CTC FISH 10 1 Setup the Chip Incision Station with blade on the top position 10 2 Place the CytoChipNano with its right side up on the indent Hold down the end of the CytoChipNano and cut the four sides of CytoChipNano with blade Do not press the middle of CytoChipNano 10 3 Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114
25. ocessed by 3pm Copyright Abnova Taiwan Corporation Authorized use only OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 15 WAbnova maman 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com CTC 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 RI 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 to a new 15 mL 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 Repeat the washing step twice 6 1 12 For CTC enumeration re suspend the cell pellet with 105 uL of WM and proceed to the protocol of CTC Capture For cell freezing and
26. riate 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 OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 1 Abnova daa 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com 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 OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 2 Abnova a 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com System Overview Background Abnova s CytoQuest CR is a non invasive system for capture enumeration isolation and retrieval of circulati
27. t Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87511888 Fax 886 2 6602 1218 12 Abnova maman 9F No 108 Jhouzih St Taipei Taiwan Tel 886 2 8751 1888 Fax 886 2 6602 1218 E mail sales abnova com Reagent Preparation N NOTE 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 KHoPOs 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 1 Triton X 100 Add 0 1 mL of Triton X 100 to 100 mL of 1X ADB e 4 PFA 4 Paraformaldehyde Aqueous Solution Dilute 20 paraformaldehyde aqueous solution with 1X PBS 4 PFA should be aliquoted and frozen at 20 C 4 PFA can be stored at 4 C for one 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 OF No 108 Jhouzih St Neihu District Taipei 114 Taiwan PP PLM A 027 Version 1 3 28 Nov 2014 Tel 886 2 87

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