Back to Top
Protocol

IsoCode Single-Cell Adaptive Immune: Human CAR-T Protocol Using AF647-Conjugated Antibodies to CD4 and CD8

  • IsoPlexis Applications Team 1
  • 1 - IsoPlexis

Jul 22, 2021

Abstract

Overview of Protocol 

Day 1: Cryopreserved CAR-T cells are thawed and cultured overnight in the presence of IL-2. Cryopreserved target cell lines are thawed, cultured, and passaged several days prior to beginning the experiment. 

Day 2: Enrichment and Antigen-Stimulation of CD8 + and/or CD4 + CAR-T cells for 20 hours. 

Day3: Staining and Loading of CAR-T cells onto IsoCode chip. 


Introduction

NOTE: This protocol outlines the standard method for thawing and culturing of human CAR-T cells only and may not be valid for other species or cell types. 

NOTE: Using stains and protocols other than the included kit surface stains and protocols might result in failed runs. Stains and staining procedures not approved by IsoPlexis will require validation prior to use. Please consider IsoPlexis’ IsoPACE™ program to assist in custom marker and protocol validation.

Safety Warnings - Read MSDS documents of all materials prior to use. - Laboratory workers should wear standard PPE, including disposable gloves, protective eyewear, and laboratory coats

 


Reagents and Equipment

Required Consumables

IsoCode Kit Components:

  • IsoCode Reagent Box (4°C)
    • 15 mL Tube A
    • 15 mL Tube B
    • 1.5 mL Tubes A/B: Cocktail A in Micro-Tube (Green Cap) and Cocktail B in Micro-Tube (Red Cap)
    • 50 mL Tubes containing Reagents 1, 2, 3, 4, 5, 6, 7, 8
    • 1 Bag of Disposable Reagent Sippers
    • Alexa Fluor 647 anti-human CD4 stain (AF647-CD4) [ordered separately]
    • Alexa Fluor 647 anti-human CD8 stain (AF647-CD8) [ordered separately]
    • K562 Cell Line Depletion Kit [ordered separately]
    • Raji Cell Line Depletion Kit [ordered separately]
  • IsoCode Chip Set (-20°C)
    • Boxes of IsoCode Chips (2 per box) to make up 4, 6, or 8 chips

 


Procedure

Before Getting Started:

1. Important Precautions

  • Read MSDS documents of all materials prior to use.
  • Working with Biohazardous Reagents: Please refer to your institute’s guidelines and obtain proper training to handle potentially biohazardous samples. It is also strongly recommended that any lab personnel handling human samples should be vaccinated against HBV if the individual does not have sufficient HBV antibody titer.
  • Additional precautions need to be taken when working with samples that potentially contain an EID agent:
    • Laboratory workers should wear standard PPE, including disposable gloves, protective eyewear, and laboratory coats.
    • Any procedure or process that cannot be conducted in the designated EID BSC should be performed while wearing gloves, gown, goggles, and a fit tested N-95 mask.
    • Work surfaces should be decontaminated on completion of work with appropriate disinfectants. This includes any surface that potentially comes in contact with the specimen (centrifuge, microscope, etc.).
    • All liquid waste produced in the processes must be treated to a final concentration of 10% bleach prior to disposal.

2. Reagents to be Prepared Before Starting

CRITICAL: Prepare 200 µL IL-2 aliquots and freeze at -20°C for no longer than 1 month. Aliquots are single use only and are to be thawed immediately prior to their usage. If there is any remaining volume in an aliquot, do not refreeze but discard. 

Protocol:

Chapter 1: Getting Started

Kit Contents:

  • IsoCode Reagent Box (4°C) 
  • IsoCode Chip Set (-20°C) 
  • Target Cell Line Depletion Kit (4°C) – 1 Kit Per 8 Samples 
    • Target Cell Depletion Beads 
    • 8 x Polystyrene Tubes 
    • *Use K562 Cell Line Depletion Beads for target cells expressing CD235a 
    • *Use Raji Cell Line Depletion Beads for target cells expressing CD19 

Chapter 2: Recovery of Cryopreserved Target Cell Lines

Materials Required:

  • Complete RPMI (37°C) 
  • 15 mL Centrifuge Tube (Target Cells) 
  • Cryopreserved Target Cell Lines 
  • Plate and/or Flask 
    • For > 10 M Cells, T75 Flask 
    • For 6-9.9 M Cells, T25 Flask
    • For < 6 M, 6 Well Plate 

All the following steps should take place in a sterile tissue culture hood.

Methods:

CRITICAL: This step should be performed at least three days prior to the recovery of CAR-T cells (Chapter 4). A week in advance is recommended. 

1. Pipette 5 mL of complete RPMI into a 15 mL centrifuge tube, labeled Target Cells. 

2. Using proper PPE, remove cells from liquid nitrogen storage and thaw cells. TIP: Be careful of contamination. 

3. Quickly move vial(s) into a water bath (37°C) to thaw. While thawing, swirl the vial in the water until a single ice crystal remains in the vial. Be sure to prevent (to the best of your ability) any of the water from the water bath from getting under the cap and into the sample. 

4. When the sample is nearly thawed, remove the vial and immediately spray vial with 70% alcohol before bringing into the hood. It is important to allow the alcohol to evaporate before opening the vial. 

5. Slowly pipette thawed cells into the 5 mL of complete RPMI in 15 mL centrifuge tube, labeled Target Cells. TIP: Insert tip into complete RPMI when pipetting, be careful to not create bubbles. 

6. Take 1 mL of the cell/complete RPMI mixture. 

7. Pipette into original thawed cell vial, rinse inside the vial with the complete RPMI to recover additional thawed cells. TIP: Insert tip into complete RPMI, be careful not to create bubbles. 

8. Draw up cell/complete RPMI mixture and pipette back into the 15 mL centrifuge tube. TIP: Insert tip into complete RPMI and pipette gently up and down. Be careful not to create bubbles. 

9. Centrifuge cells for 10 minutes at 300 rcf. 

10. Remove cells from centrifuge, check for cell pellet. 

11. Aspirate supernatant. TIP: Be careful not to aspirate the cell pellet. 

  • Use pipette to remove last bit of supernatant. 

12. Resuspend in 1 mL of fresh complete RPMI. TIP: Make sure to mix well. Be careful not to create bubbles or leave clumps. 

13. Slowly add complete RPMI to a final concentration of 1 x 106 cells/mL. 

14. Mix well 5 times with serological pipette. TIP: Be careful not to create bubbles. 

15. Transfer cell suspension to flask or plate. TIP: Slowly pipette down the side of the flask as to not create bubbles

16. Spread out cell suspension by rocking flask or plate carefully to fully cover the bottom of the container. TIP: Be careful to not make bubbles. 

17. Incubate at 37°C, 5% CO2. 

18. Passage cells every few days, depending on the requirements for specific cell type.

Chapter 3: Culture of Target Cells 

Materials Required:

  • Complete RPMI (37°C) 
  • Incubated Cells from Chapter 2 
  • 15 mL Centrifuge Tube 
  • Lo-Bind Microcentrifuge Tube for Cell Count 
  • T75 Flask 

All the following steps should take place in a sterile tissue culture hood. 

Methods:

1. Transfer cells from flask or plate into 15 mL centrifuge tube. TIP: Be careful not to create bubbles. 

2. Add complete RPMI to flask or plate and rinse 5 times. TIP: Make sure to spread out the complete RPMI to gather maximum number of cells. 

  • For T75 Flask add 3 mL 
  • For T25 Flask add 2 mL 
  • For 6 Well Plate add 1 mL 

3. Transfer cell/complete RPMI mixture to the 15 mL centrifuge tube. 

4. Mix well 5 times with 10 mL serological pipette. TIP: Be careful not to create bubbles. 

5. Take a 10 µL aliquot of your cells and transfer to a Lo-Bind Microcentrifuge Tube for cell counting. CRITICAL: See Appendix D1 for cell counting instructions. 

6. Centrifuge cells for 10 minutes at 300 rcf. While cells are centrifuging, use hemocytometer to count cells. CRITICAL: See Appendix D1 for cell counting instructions. 

7. Remove cells from centrifuge, check for cell pellet. 

8. Aspirate supernatant. TIP: Be careful not to aspirate the cell pellet.

  • Use pipette to remove last bit of supernatant. 

9. Resuspend target line cells in 1 mL of complete RPMI. TIP: Make sure to mix well. Be careful not to create bubbles or leave clumps. 

10. Slowly add complete RPMI to a final concentration of 1 x 106 cells/mL. 

11. Mix with serological pipette by gently pipetting up and down 5 times. TIP: Be careful not to create bubbles. 

12. Transfer cell suspension to flask. TIP: Slowly pipette down the side of the flask as to not create bubbles. 

13. Spread out cell suspension by rocking flask carefully to fully cover the bottom of the flask. TIP: Be careful to not make bubbles. 

14. Incubate at 37°C, 5% CO2 and passage the target and control cells as needed over the next few days. 

CRITICAL: Passage sufficient numbers of target and control cells to perform assay at a ratio of 1:2 CAR-T cells to targets.

Chapter 4: Recovery of Cryopreserved CAR-T Cells

Materials Required:

  • Complete RPMI (37°C) 
  • Recombinant IL-2 at 1 µg/mL (-20°C) 
  • 15 mL Centrifuge Tube (Thawed CAR-T) 
  • Cryopreserved CAR-T Cells 
  • Plate and/or Flask 
    • For > 10 M Cells, T75 Flask 
    • For 6-9.9 M Cells, T25 Flask 
    • For < 6 M, 6 Well Plate 

All the following steps should take place in a sterile tissue culture hood. 

Methods:

1. Pipette 5 mL of complete RPMI into a 15 mL centrifuge tube, labeled Thawed CAR-T. 

2. Using proper PPE, remove cells from liquid nitrogen storage and thaw cells. TIP: Be careful of contamination

3. Quickly move vials into a water bath (37°C) to thaw. While thawing, swirl the vial in the water until a single ice crystal remains in the vial. Be sure to prevent (to the best of your ability) any of the water from the water bath from getting under the cap and into the sample. 

4. When the sample is nearly thawed, remove the vial and immediately spray vial with 70% alcohol before bringing into the hood. It is important to allow the alcohol to evaporate before opening the vial. 

5. Slowly pipette thawed cells into 5 mL of complete RPMI in 15 mL centrifuge tube, labeled Thawed CAR-T. TIP: Insert tip into complete RPMI when pipetting, be careful not to create bubbles. 

6. Take 1 mL of the cell/complete RPMI mixture. 

7. Pipette into original thawed cell vial, rinse inside the vial with the complete RPMI to recover additional thawed cells. TIP: Insert tip into complete RPMI, be careful not to create bubbles. 

8. Draw up cell/complete RPMI mixture and pipette back into the 15 mL centrifuge tube. TIP: Insert tip into complete RPMI and pipette gently up and down. Be careful not to create bubbles. 

9. Centrifuge cells for 10 minutes at 300 rcf. 

10. While the cells are centrifuging, take the IL-2 (1µg/mL) out from -20°C and thaw at room temperature. 

CRITICAL: Use IL-2 aliquot that has been frozen at -20°C for less than a month. Do not use IL-2 that has been previously thawed. 

11. Remove cells from centrifuge, check for cell pellet. 

12. Aspirate supernatant. TIP: Be careful not to aspirate the cell pellet. 

  • Use pipette to remove last bit of supernatant. 

13. Resuspend in 1 mL of fresh complete RPMI. TIP: Make sure to mix well. Be careful not to create bubbles or leave clumps. 

14. Slowly add complete RPMI to a final concentration of 1 x 106 cells/mL. 

15. Mix thawed IL-2 thoroughly by carefully pipetting up and down. 

16. Dilute 100 µL of 1 µg/mL IL-2 per 10 mL of cell suspension to a final concentration of 10 ng/mL. CRITICAL: Discard thawed IL-2 aliquot if there is any volume remaining. IL-2 must only be thawed once. 

17. Mix with serological pipette by gently pipetting up and down 5 times. TIP: Be careful to not create bubbles. 

18. Transfer cell suspension to flask or plate. TIP: Slowly pipette down the side of the flask as to not create bubbles. 

19. Spread out cell suspension by rocking flask or plate carefully to fully cover the bottom of the container. TIP: Be careful to not make bubbles. 

20. Move to incubator for overnight recovery at 37°C, 5% CO2. 

Chapter 5: Pre-Sample Enrichment 

Materials Required:

  • Complete RPMI (37°C) 
  • 15 mL Centrifuge Tube 
  • Overnight Recovered CAR-T Cells from Chapter 4 
  • Lo-Bind Microcentrifuge Tube 

All the following steps should take place in a sterile tissue culture hood. 

Methods:

1. Transfer cells from flask or plate into 15 mL centrifuge tube. TIP: Be careful not to create bubbles. 

2. Add complete RPMI to flask and rinse 5 times. TIP: Make sure to spread out the complete RPMI to gather maximum number of cells. 

  • For T75 Flask add 3 mL
  • For T25 Flask add 2 mL 
  • For 6 Well Plate add 1 mL 

3. Transfer cell/complete RPMI mixture to the 15 mL centrifuge tube. 

4. Mix with 10 mL serological pipette by gently pipetting up and down 5 times. TIP: Be careful not to create bubbles.

5. Take a 10 µL aliquot of your cells and transfer to a Lo-Bind Microcentrifuge Tube for cell counting. CRITICAL: See Appendix D1 for cell counting instructions.

6. Centrifuge cells for 10 minutes at 300 rcf. While cells are centrifuging, use hemocytometer to count cells. CRITICAL: See Appendix D1 for cell counting instructions. 

CRITICAL: If cells are less than 80% viable, proceed to Appendix D2 Dead Cell Depletion Protocol using Ficoll. 

7. Proceed immediately to next chapter. 

Chapter 6: CD8 Sample Enrichment

 Materials Required:

  • Complete RPMI (37°C) 
  • RoboSep Buffer (4°C) 
  • Miltenyi CD8 Microbeads, Human, 2 mL (4°C) 
  • MACS LS Column 
  • Prepared Cells from Chapter 5 
  • Enrichment Kit: 
    • MACS Metal Plate/Magnet Kit 
    • 3 x 15 mL Centrifuge Tubes (Discard, Flow Through (CD4), CD8 Fraction) 

All the following steps should take place in a sterile tissue culture hood. 

Methods:

1. Remove cells from centrifuge, check for cell pellet. 

2. Aspirate supernatant. TIP: Be careful not to aspirate the cell pellet.

  • Use pipette to aspirate remaining supernatant. 

CRITICAL: For every 1 x 107 cells, resuspend in 80 µL RoboSep (4°C) and 20 µL of CD8 beads (4°C). 

3. Add 80 µL of cold RoboSep to 15 mL centrifuge tube containing 1 x 107 or fewer cells. 

4. Vortex Miltenyi CD8 Microbeads at a slow speed for 10 seconds. 

5. Add 20 µL of Miltenyi CD8 Microbeads and mix well by gently pipetting up and down 5 times. 

TIP: Make sure to eliminate clumps so that beads are evenly distributed among cells. Be careful not to create bubbles. 

6. Incubate in refrigerator (4°C) for 15 minutes. 

7. After 15 minutes, add 2 mL of cold RoboSep. TIP: Not necessary to mix for this step

8. Centrifuge cells for 10 minutes at 300 rcf.

TIP: Keep RoboSep in refrigerator during enrichment process. 

9. Set up MACS sorting by setting metal plate in tissue culture hood and placing magnet on metal plate. Place LS column in magnet with wings facing out and align the 15 mL centrifuge tube labeled “Discard” under the LS column. CRITICAL: LS Column should not touch the tubes. 

10. After cells are centrifuged, check for cell pellet and continue with MACS separation. 

11. Aspirate RoboSep from cell pellet. TIP: Since it is a small volume, use pipette for this step to prevent accidental aspiration of the cell pellet. 

12. For 1 x 108 or fewer cells, resuspend with 500 µL of cold RoboSep.

  • Mix well to resuspend by gently pipetting up and down 5 times. TIP: Make sure to pipette around the tube to ensure there are no clumps or bubbles. 

CRITICAL: Be careful not to let column dry out. Make sure not to add liquid when there is already liquid in the LS Column. 

13. Starting with the LS column over the “Discard” tube, add 3 mL of cold RoboSep to LS Column. CRITICAL: Be careful not to create bubbles or touch sides of LS column. Let all the RoboSep flow through before moving on to next step. As a reminder, be careful to not let the column dry out. 

14. Unscrew and keep cap for “Flow Through (CD4)” tube. NOTE: This is in preparation for next step to ensure the column does not dry out during the transition. 

15. When last drop falls through to ”Discard” tube, move the rack over so the LS column is over the flow through tube. CRITICAL: Be careful not to let column dry. If there is one drop remaining that will not fall, move on to next step. 

16. Increase volume of pipette to 800 µL to ensure all 500 µL of the cell suspension is drawn up. 

17. Mix cell suspension by gently pipetting up and down 5 times. NOTE: This ensures that the cells are evenly dispersed after sitting. 

18. Draw up all 500 µL of cell suspension and pipette carefully into the center of the LS column without touching sides. 

19. Wash 3 times with 3 mL of cold RoboSep. 

  • First wash: Rinse inside walls of cell suspension tube with 3 mL of RoboSep before transferring the mixture to LS Column. NOTE: This is to retrieve any cells that have been left behind. 
    • Pipette all the mixture into LS Column after last drop passes through or does not fall from step 18. CRITICAL: Be careful not to let LS Column dry out or allow pipette to touch sides. 
  • Second wash: Add 3 mL of RoboSep into LS Column after last drop passes through or does not fall. CRITICAL: Be careful not to let LS Column dry out or allow pipette to touch sides. 
  • Third wash: Add 3 mL of RoboSep into LS Column after last drop passes through or does not fall. CRITICAL: Be careful not to let LS Column dry out or allow pipette to touch sides. 

20. After last drop of the third wash passes through or does not fall, remove the LS Column carefully from the magnet, and place carefully on the tube labeled for “CD8 fraction”. 

21. Cap the “Flow Through (CD4)” tube. This will be used for CD4 CAR-T cell subset in Cell Stimulation (Chapter 7). Do not discard. 

22. Add 5 mL of cold RoboSep to the LS column. CRITICAL: Be careful not to touch the sides

23. Take plunger, smoothly push down on the plunger to push the RoboSep buffer through the LS Column. TIP: Lift up at the end of the plunging action so that the liquid does not splash back onto the LS Column tip. 

24. Set LS Column back on the “CD8 fraction” tube. 

25. Loosen up plunger. Remove plunger briefly from column and hold in one hand. CRITICAL: Do not allow the plunger to interact with external contaminants. It will be used for one more step. 

26. Add another 4 mL of cold RoboSep to the LS Column. 

27. Take plunger, smoothly push down on the plunger to push the RoboSep buffer through the LS Column. TIP: Lift up at the end of the plunging action so that the liquid does not splash back onto the LS Column tip. 

28. Discard LS Column and plunger. 

29. Centrifuge “CD8 Fraction” tube and “Flow Through (CD4)” tube for 10 minutes at 300 rcf. 

30. Remove cells from centrifuge, check for cell pellets. 

31. Aspirate RoboSep buffer from “CD8 fraction” and “Flow Through (CD4)” tubes. TIP: Be careful to not aspirate cell pellet. 

32. Use pipette to aspirate the remaining supernatant from each tube. TIP: Be careful to not aspirate cell pellet. 

33. Gently resuspend in 500 µL of complete RPMI.

  • Mix well to resuspend by gently pipetting up and down 5 times. TIP: Make sure there are no clumps or bubbles. 

34. Take a 10 µL aliquot of cell fractions and transfer to a Lo-Bind Microcentrifuge Tube for cell counting. CRITICAL: See Appendix D1 for cell counting instructions. 

35. Centrifuge cells for 10 minutes at 300 rcf. While cells are spinning down, use hemocytometer to get cell counts. CRITICAL: See Appendix D1 for cell counting instructions. 

36. Remove cells from centrifuge, check for cell pellets. 

37. Aspirate supernatant with a pipette. TIP: Be careful not to aspirate the cells. 

38. Gently resuspend cell fractions at a density of 1 x 106 cells/mL in complete RPMI. 

  • Mix well to resuspend by gently pipetting up and down 5 times. TIP: Make sure to pipette around the tube to ensure there are no clumps or bubbles.

 39. Incubate cell suspensions at 37°C 5% CO2 until Cell Stimulation (Chapter 7).

Chapter 7: Cell Stimulation 

Materials Required:

  • Complete RPMI (37°C) 
  • 2 x 15 mL Centrifuge Tubes 
  • 96 Well Plate U-Bottom 
  • CAR-T Specific Target Cell Culture(s) from Chapter 2 
  • CD8 Fraction
  • Flow Through (CD4) 
  • 2 x Lo-Bind Microcentrifuge Tubes 

All the following steps should take place in a sterile tissue culture hood. 

Methods:

1. Mix target cell line(s) gently up and down using a pipette. TIP: Be careful not to create bubbles. 

2. Transfer target cells from flask into 15 mL Centrifuge Tube. TIP: Be careful not to create bubbles. 

3. Add complete RPMI to flask and rinse 5 times. TIP: Make sure to spread out the complete RPMI to gather maximum number of cells.

  • For T75 Flask add 3 mL
  • For T25 Flask add 2 mL
  • For 6 Well Plate add 1 mL

4. Transfer target cells/complete RPMI mixture to the 15 mL Centrifuge Tube. TIP: Be careful not to create bubbles. 

5. Take a 10 µL aliquot of target cells and transfer to a Lo-Bind Microcentrifuge Tube for cell counting. CRITICAL: See Appendix D1 for cell counting instructions.

6. Centrifuge cells for 10 minutes at 300 rcf. While cells are spinning down, use hemocytometer to get cell counts. CRITICAL: See Appendix D1 for cell counting instructions. 

CRITICAL: If cells are less than 80% viable, proceed to Appendix D2 Dead Cell Depletion Protocol using Ficoll. 

7. Remove cells from centrifuge, check for cell pellet. 

8. Aspirate supernatant. TIP: Be careful not to aspirate the cells.

  • Use pipette to aspirate remaining supernatant. 

9. Resuspend target cells with complete RPMI to a cell concentration of 2 x 106 cells/mL. TIP: Resuspend as thoroughly as possible, but gently

10. Using a P100 pipette, add 100 µL of CAR-T specific target cells to desired number of wells in the 96 Well UBottom Plate. NOTE: Concentration is 2 x 106 cells/mL.

CRITICAL: Use a 1:1 volume ratio for the overall density ratio of 1 CAR-T cell to 2 CAR-T specific target cells. Volume in each well should be 200 µL. 

11. Add 100 µL of the CD8 CAR-T cells to the well(s) containing the CAR-T specific target cells in the 96 Well UBottom Plate. NOTE: Concentration is 1 x 106 cells/mL.

  • Mix cell suspension thoroughly. TIP: Be careful not to create bubbles. Pipette thoroughly, but gently. 

12. Repeat step 11 with the CD4 CAR-T cells. 

CRITICAL: Be careful not to create bubbles. This will maximize even stimulation of cell suspension. 

13. Incubate 96 Well Plate for 20 hours at 37°C, 5% CO2. 

Chapter 8: Target Cell Depletion 

Target Cell Depletion Kits:

Materials Required:

  • 96 Well Plate Containing CD8 CAR-T CoCultures and CD4 CAR-T Co-Cultures 
  • Sterile 1X PBS (room temperature) 
  • EasySepTM Magnets 
  • 2 x 15 mL Centrifuge Tubes 
  • 4 x Lo-Bind Microcentrifuge Tubes 
  • 2 x 5 mL Polystyrene Tubes 
  • Target Cell Line Depletion Beads (4°C) 

All the following steps should take place in a sterile tissue culture hood. 

Methods:

CRITICAL: Use the K562 Cell Line Depletion Beads for target cells expressing CD235a. Use the Raji Cell Line Depletion Beads for target cells expressing CD19. 

1. Resuspend CD8 CAR-T cells by pipetting up and down before transferring into a Lo-Bind Microcentrifuge Tube. TIP: Make sure to mix well. Be careful not to create bubbles or leave clumps. 

2. Repeat step 1 using CD4 CAR-T cells. 

3. Centrifuge cells for 10 minutes at 300 rcf. 

4. Remove cells from centrifuge, check for cell pellet. 

5. *Aspirate supernatant with a pipette. TIP: Be careful not to aspirate the cell pellets. *NOTE: Supernatants may be stored at -80°C for population assay. 

6. Remove depletion beads from 4°C and resuspend beads using a P1000 pipette. TIP: Make sure to eliminate clumps so that beads are evenly distributed among cells. Be careful not to create bubbles. 

7. CRITICAL: For every 3 x 105 cells, add 50 µL of depletion beads (e.g., antiCD235a-conjugated beads for K562 cell depletion) to each cell pellet and return remainder of beads to 4°C. 

  • Mix well to resuspend. TIP: Make sure to pipette around the tube to ensure there are no clumps or bubbles

8. Incubate for 10 minutes at room temperature. 

9. Gently mix with a P100 pipette to ensure beads are kept in suspension. CRITICAL: Resuspend every 2 minutes to keep mixture in suspension. 

10. After the incubation, add 950 µL of PBS to the CD8 CAR-T cells. Repeat step using CD4 CAR-T cells. 

11. Gently pipette up and down to resuspend. TIP: Make sure to mix well. Be careful not to create bubbles or leave clumps. 

12. Transfer CD8 CAR-T mixture from Lo-Bind Microcentrifuge Tube to a pre-labeled 5 mL polystyrene tube. Repeat step using CD4 CAR-T cells. 

13. Place 5 mL polystyrene tubes in EasySepTM magnets for 2 minutes. 

14. After 2 minutes, keep 5 mL polystyrene tubes attached to the magnets and forcefully decant cells into 15 mL centrifuge tubes. CRITICAL: Be careful not to disrupt the 5 mL Polystyrene Tube from magnet. 

15. Remove 5 mL polystyrene tubes from the EasySepTM magnets. 

16. Add 1 mL of PBS to the empty Lo-Bind Microcentrifuge Tubes from Step 12 to recover any remaining material. 

17. Transfer contents to corresponding 5 mL polystyrene tubes. NOTE: Make sure to rinse the walls of 5 mL Polystyrene Tubes well. 

18. Place 5 mL polystyrene tubes in an EasySepTM magnets for 2 minutes. 

19. After 2 minutes, keep 5 mL polystyrene tubes attached to the magnets and forcefully decant cells into 15 mL Centrifuge Tubes. CRITICAL: Be careful not to disrupt the 5 mL Polystyrene Tube from magnet. NOTE: Pool target cell depleted T cell suspensions from the same sample into one 15 mL centrifuge tube. 

20. Centrifuge CAR-T cells for 10 minutes at 300 rcf. 

21. Remove cells from centrifuge, check for cell pellets. 

22. Aspirate supernatant. TIP: Be careful not to aspirate cell pellets. a. Use pipette to aspirate remainder of supernatant. 

23. Gently resuspend in 500 µL of complete RPMI. 

  • Mix well to resuspend by gently pipetting up and down 5 times. TIP: Make sure to pipette around the tube to ensure there are no clumps or bubbles. 

24. Increase volume of pipette to 800 µL to ensure all 500 µL of the cell suspension is drawn up. 

25. Draw up all 500 µL of target depleted CD8 CAR-T cells and pipette into a Lo-Bind Microcentrifuge Tube. Repeat step with CD4 CAR-T cells. 

26. Centrifuge CAR-T cells for 10 minutes at 300 rcf. 

Chapter 9: Chip Thawing

Materials Required:

  • IsoCode Chips in Vacuum Sealed Bag (-20°C) 

Methods: 

1. Take vacuum sealed bag containing IsoCode chips from -20°C. CRITICAL: Chips must stay sealed until Chip Loading (Chapter 11). 

2. Place on a bench to thaw at ambient temperature 30-60 minutes prior to use. 

3. While chips thaw, prepare liquid reagents and attach all reagent tubes to IsoLight. Refer to the IsoLight System Guide for detailed instructions. 

Chapter 10: Cell Staining 

Materials Required:

  • Complete RPMI (37°C) 
  • AF647 anti-human CD8 (4°C) 
  • AF647 anti-human CD4 (4°C) 
  • CD8 CAR-T Cells 
  • CD4 CAR-T Cells 
  • 2 x Lo-Bind Microcentrifuge Tubes 

All the following steps should take place in a sterile tissue culture hood. 

Methods:

1. Remove cells from centrifuge, check for cell pellets. 

2. Aspirate supernatant with a pipette. TIP: Be careful not to aspirate the cell pellets. 

3. Gently resuspend in 18 µL of complete RPMI.

  • Mix well to resuspend by gently pipetting up and down 5 times. TIP: Make sure to pipette around the tube to ensure there are no clumps or bubbles.

4. Centrifuge tubes of AF647 anti-human CD8 stain & AF647 anti-human CD4 stain in a micro centrifuge for 10 seconds to collect the stain at the bottom of the tubes. 

5. Add 2 µL (1:10 final dilution) of AF647 anti-human CD8 to CD8 CAR-T cells. Mix gently by pipetting up and down. CRITICAL: Be sure to use appropriate stain for each cell subset. 

6. Add 2 µL (1:10 final dilution) of AF647 anti-human CD4 to CD4 CAR-T cells. Mix gently by pipetting up and down. CRITICAL: Be sure to use appropriate stain for each cell subset. 

7. Incubate stained CD8 CAR-T cells and CD4 CAR-T cells for 20 minutes at room temperature in the dark. 

8. After 20 minutes, add 1 mL of complete RPMI to the stained cells. TIP: Mix gently, be careful not to create bubbles. 

9. Centrifuge stained cells for 10 minutes at 300 rcf. 

10. Remove cells from centrifuge, check for cell pellets. 

11. Aspirate supernatant with a pipette. TIP: Be careful not to aspirate the cell pellets. 

12. Gently resuspend in 500 µL of complete RPMI. 

  • Mix well to resuspend by gently pipetting up and down 5 times. TIP: Make sure to pipette around the tube to ensure there are no clumps or bubbles. 

13. Take a 10 µL aliquot of stained CD8 CAR-T cells and stained CD4 CAR-T cells, transfer to a Lo-Bind Microcentrifuge Tube for cell counting. CRITICAL: See our Cell Quantification & Viability Protocol for instructions. 

14. Centrifuge stained cells for 10 minutes at 300 rcf. While cells are spinning down, use hemocytometer to get cell counts. CRITICAL: See Appendix D1 for cell counting instructions. 

15. Remove cells from centrifuge, check for cell pellets. 

16. Aspirate supernatant with a pipette. TIP: Be careful not to aspirate the cell pellets. 

17. Gently resuspend CAR-T cells at 1 x 106 cells/mL in complete RPMI.

  • Mix well to resuspend. TIP: Make sure to pipette around the tube to ensure there are no clumps or bubbles. 

18. Incubate stained CD8 CAR-T cells and CD4 CAR-T cells at 37°C 5% CO2 until Chip Loading (Chapter 11).

Chapter 11: Chip Loading 

Materials Required (Pre-prepared):

  • Pre-Thawed IsoCode Chips in Vacuum Sealed Bag from Chapter 9 
  • CD8 CAR-T Cells at 1 x 106 Cells/mL 
  • CD4 CAR-T Cells at 1 x 106 Cells/mL 

Methods:

 NOTE: CD4 CAR-T cells and CD8 CAR-T cells should be loaded on separate chips as the stain will not distinguish between the two types. 

1. Remove IsoCode chips from vacuum sealed bag and place on a flat surface. CRITICAL: Keep protective blue film on bottom. 

2. Resuspend CD8 CAR-T cells by pipetting up and down. Pipette 30 µL of cell suspension into IsoCode chip. CRITICAL: Be careful not to create bubbles. Insert pipette tip vertically into inlet port until tip lightly touches bottom, and slowly pipette 30 µL into inlet port. Be careful not to eject second step of pipette–it will cause bubbles. 

3. Resuspend CD4 CAR-T cells by pipetting up and down. Pipette 30 µL of cell suspension into IsoCode chip. CRITICAL: Be careful not to create bubbles. Insert pipette tip vertically into inlet port until tip lightly touches bottom, and slowly pipette 30 µL into inlet port. Be careful not to eject second step of pipette–it will cause bubbles. 

4. Let IsoCode chips sit for one minute on a flat surface. 

5. Check bottom of chip to ensure liquid has entered the chip. TIP: If liquid has not flowed, tap IsoCode chip on flat surface lightly. 

6. When inserting IsoCode chip into IsoLight, make sure barcode is facing up and towards you with the magnet facing the IsoLight. Take the blue film off while inserting each IsoCode chip into the IsoLight. 

NOTE: Please refer to the loading instructions of the IsoLight instrument for details. 

 

 

 

 


Notes and Comments

Appendix 

D1 Protocol: 

  • Cell Quantification & Viability Hemocytometer
  • 10 µL aliquot of cells
  • Trypan Blue

NOTE: Automated cell counters can be used in this protocol EXCEPT prior to loading cells on chip due to spectral overlap of the stains. Manual cell counting is required prior to loading on the chip. 

1. Using a P10 pipette, add equal volume of Trypan blue solution to 10 µL of sample. Mix gently to resuspend. TIP: Make sure to pipette around the tube to ensure there are no clumps or bubbles.

2. Load onto hemocytometer. CRITICAL: Be careful not to overfill or create bubbles. 

3. Count and record viable (clear) and dead cells (blue) of all four 16-square corners. CRITICAL: If more than 200 cells/16 squares were counted, repeat count using a 1:5 or 1:10 dilution with 1X PBS or complete RPMI using a fresh sample aliquot. 

4. Calculate the concentration of cells as follows:

  • Concentration (cells/mL) = Average per square cell count x 104 x dilution factor

5. Calculate the number of cells as follows: 

  • Number of cells = Cell concentration (cells/mL) from D.1.3 x total volume of cell suspension (mL)

6. Calculate percent viable cells: 

  • % Viable cells = 100 x number of viable cells / [number of viable cells + number of dead cells]

D2 Protocol: Dead Cell Removal Using Ficoll 

Materials Required: 

  • Complete RPMI (37°C) 
  • Cells (Minimum 3 x 106 ) 
  • 2 x 15 mL Centrifuge Tubes 
  • Ficoll Paque (room temperature) 

CRITICAL: It is recommended to start this protocol with a minimum of 3 x 106 total cells. 

1. Carefully add 6 mL of Ficoll to the bottom of the required amount of 15 mL centrifuge tube(s) prior to harvesting cultures. 

2. Centrifuge cells for 10 minutes at 300 rcf. 

3. Remove cells from centrifuge, check for cell pellet. 

4. Aspirate supernatant. TIP: Be careful not to aspirate cell pellet. a. Use pipette to aspirate remaining supernatant. 

5. Resuspend the pellet(s) in 7 mL of complete RPMI. TIP: Be careful not to create bubbles. 

  • Mix well to resuspend by gently pipetting up and down 5 times. TIP: Make sure to pipette around the tube to ensure there are no clumps or bubbles

CRITICAL: Do not use more than 1 x 107 cells of your suspension per Ficoll tube. 

6. Add the cell suspension(s) VERY SLOWLY to the tube(s) containing Ficoll. CRITICAL: Place the tip of your pipette on the wall of the tube, close to the Ficoll layer. Add cell suspension VERY SLOWLY. 

CRITICAL: This step must be done carefully and slowly to avoid mixing of the layers. 

7. Centrifuge tubes for 20 minutes at 300 rcf without break or acceleration. CRITICAL: Turn acceleration and brakes off to preserve the density layers established during centrifugation. 

8. While cells centrifuge, prepare appropriate amount of 15 mL centrifuge tube(s) containing 6 mL of complete RPMI. 

9. Remove cells from centrifuge, check for cloudy layer which are the viable cells. 

10. Aspirate a small volume of the supernatant. CRITICAL: Be careful not to aspirate cloudy layer containing cells. 

11. Using a P1000 pipette, collect the viable cells by recovering the cloudy layer between Ficoll and complete RPMI media. 

12. Transfer cells into the 15 mL centrifuge tube(s) containing complete RPMI. 

13. Aliquot 10 µL of cell/complete RPMI mixture(s) into a Lo-Bind Microcentrifuge Tube(s) and proceed to cell count. CRITICAL: See Appendix D1 for cell counting instructions. 

For troubleshooting, see pages 26-27 on the PDF of the protocol, or contact Support at 475-221-8402 & support@isoplexis.com with specific troubleshooting questions. 


References

IsoPlexis