Nov 17, 2023

Public workspaceCAR T cell characterization by flow cytometry

DOI
dx.doi.org/10.17504/protocols.io.kqdg3xyp1g25/v1
  • 1Duke University
  • Andrea R Daniel: This protocols was adapted form the M. Brown lab at Duke University
  • Gersbach Lab
Andrea R Daniel
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DOI: dx.doi.org/10.17504/protocols.io.kqdg3xyp1g25/v1
Protocol Citation: Sean R. McCutcheon, Adam M. Swartz, Michael C. Brown, Alejandro Barrera, Christian McRoberts Amador, Keith Siklenka, Lucas Humayun, Maria A. ter Weele, James M. Isaacs, Andrea R Daniel, Timothy E. Reddy, Andrew S. Allen, Smita K. Nair, Scott J. Antonia, Charles A. Gersbach 2023. CAR T cell characterization by flow cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3xyp1g25/v1
Manuscript citation:
Transcriptional and epigenetic regulators of human CD8+ T cell function identified through orthogonal CRISPR screens.McCutcheon SR, Swartz AM, Brown MC, Barrera A, McRoberts Amador C, Siklenka K, Humayun L, Ter Weele MA, Isaacs JM, Reddy TE, Allen AS, Nair SK, Antonia SJ, Gersbach CA.Nat Genet. 2023 Nov 9. doi: 10.1038/s41588-023-01554-0. Online ahead of print.PMID: 37945901
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 17, 2023
Last Modified: November 17, 2023
Protocol Integer ID: 91120
Keywords: CAR T cells, flow cytometry
Funders Acknowledgements:
NIH
Grant ID: HG012053
Abstract
Flow cytometry of tumor tissue and blood from mice with human tumors. Mice bearing HCC1954 tumors were euthanized at days 3 and 19 post CAR T cel delivery. This protocol describes methods for preparing input CAR T cells and tumor infiltrating CAR T cells for phenotypic characterization of by flow cytometry. Data collection can be performed using a Fortessa X 20 and analyzed using Flow Jo V10.8.1.
Materials
Antibodies Table
Antibody TargetFluorophore/SequenceCloneIsotypeDilutionApplicationManufacturerCatalog #Notes
CD2PERPA-2.10Mouse / IgG1, kappa1:50Flow cytometryThermo12-0029-42-
B2MPEA17082AMouse IgG1, κ1:50Flow cytometryBiolegend395704-
IL2RAPE-Cy7BC96Mouse / IgG1, kappa1:50Flow cytometryThermo25-0259-42-
EGFRbv-421EGFR.1Mouse IgG2b, κ1:50Flow cytometryBD Biosciences742602-
CCR7FITC150503Mouse IgG2a1:100Flow cytometryBD Biosciences561271Stain at 37C
CD8bv-421HIT8aMouse IgG1, κ1:50Flow cytometryBD Biosciences740078-
IL7RAPE-Cy5eBioRDR5Mouse / IgG1, kappa1:100Flow cytometryThermo15-1278-42-
LAG3PE3DS223HMouse / IgG1, kappa1:50Flow cytometryThermo12-2239-42-
TIM3PE-Cy5F38-2E2Mouse IgG1, κ1:50Flow cytometryBiolegend345052-
TIGITPerCP-eFluor710MBSA43Mouse / IgG1, kappa1:50Flow cytometryThermo46-9500-42-
PD1PE-Cy7EH12.1Mouse IgG1, κ1:100Flow cytometryBD Biosciences561272-
Myc-tagAlexa Fluor 6479B11Mouse IgG2a1:50Flow cytometryCell Signaling Technology2233S-
Thy1.1PEOX-7Mouse IgG1, κ1:300Flow cytometryStemCell Technologies60024PE-
Note: Antibodies below were used for in vivo TIL characterization experiment (more details in Supplemental Methods 4)
CD3BUV737UCHT1Mouse IgG1 kappa1:100Flow cytometryBD Biosciences612750
CD8BUV395RPA-T8Mouse IgG1 kappa1:100Flow cytometryBD Biosciences563795
TIGITBV605A15153GMouse IgG2a, kappa1:100Flow cytometryBiolegend372712
LAG3BV78511C3C65Mouse IgG1, Kappa1:100Flow cytometryBiolegend369322
CD127PERCP-Cy5.5A019D5Mouse IgG1, Kappa1:100Flow cytometryBiolegend351322
PD1BV711EH12.2GH7Mouse IgG1 kappa1:100Flow cytometryBiolegend329928
TIM3PE-Cy5F38-2E2Mouse IgG1, Kappa1:100Flow cytometryBiolegend345052
Granzyme BPE-Cy7QA16A02Mouse IgG1 kappa1:100Flow cytometryBiolegend372214
TCF1BV421S33-966Mouse IgG1 kappa1:100Flow cytometryBD Biosciences566692
Ki-67BV510Ki-67Mouse IgG1 kappa1:100Flow cytometryBiolegend350518
IFN-gPEB27Mouse IgG1 kappa1:100Flow cytometryBiolegend506507
CD39PE/Dazzle-594A1Mouse IgG1, Kappa1:100Flow cytometryBiolegend328224
CD56BV6055.1H11Mouse IgG1 kappa1:100Flow cytometryBiolegend362538
CD45ROBV786UCHL1Mouse IgG2a, kappa1:100Flow cytometryBiolegend304234
CD45RAPE-Cy5HI100Mouse IgG2b, kappa1:100Flow cytometryBiolegend304110
CD28PE-Cy7S20013BMouse IgG1, Kappa1:100Flow cytometryBiolegend377812
CCR7BV711G043H7Mouse IgG2a, kappa1:100Flow cytometryBiolegend353228
CD62LBV510DREG-56Mouse IgG1, Kappa1:100Flow cytometryBiolegend304844
CTLA4BV421BNI3Mouse IgG2a, kappa1:100Flow cytometryBiolegend369606
TbetPERCP-Cy5.54B10Mouse IgG1, Kappa1:100Flow cytometryBiolegend644806
EOMEsPEX4-83Mouse IgG1 kappa1:100Flow cytometryBD Biosciences566749
CD45FITCHI30Mouse IgG1, Kappa1:100Flow cytometryBiolegend304054
CXCR3BV711G025H7Mouse IgG1, Kappa1:100Flow cytometryBiolegend353732
TNFBV605MAb11Mouse IgG1, Kappa1:100Flow cytometryBiolegend502936
ID2PE-Cy7ILCID2Mouse IgG1, Kappa1:100Flow cytometryThermo-Fisher 25-9475-82
GATA3BV42116E10A23Mouse IgG2b, kappa1:100Flow cytometryBiolegend653814
IRF4PERCP-Cy5.5IRF4.3E4Rat IgG1, Kappa1:100Flow cytometryBiolegend646416
ID3PES30-778Mouse IgG1, Kappa1:100Flow cytometryBD Biosciences564564
CD4BV510OKT4Mouse IgG2b, kappa1:100Flow cytometryBiolegend317444
Materials Table
NameManufacturer
RBC lysis bufferSigma
RPMI-1640 mediumGibco
LiberaseSigma-Aldrich
DNaseIRoche
70mm cell strainerOlympus Plastics
PBSGibco
Zombie NIRBiolegend
FBSSigma
Tru-stain Fc blockBiolegend
CAR T Panels Table
HUMAN Panel 1 mainHUMAN panel 2 Human Panel 3Antigenflurophore
GFPCD45GFPCD45GFPCD45Zombie live/deadAPCy7
MYCAPCMYCAPCMYCAPCCD3BUV737
Live DeadAPCcy7Live DeadAPCcy7Live DeadAPCcy7CD8BUV395
CD3BUV737CD3BUV737CD3BUV737CD4BV510
CD8BUV395CD8BUV395CD8BUV395TiGITBV605
TIGITBV605CD39PECF594TNFBV605LAG3BV786
LAG3BV786CD56BV605LAG3BV786PD1BV711
CD127PERCPcy5.5CD45ROBV786TIM3PEcy5TIM3PEcy5
Granzyme BPecy7CD45RAPEcy5ID2PeCy7TCF1BV421
PD1BV711CD28PeCy7CXCR3BV711Ki67BV510
TIM3PEcy5CCR7BV711CD4BV510ID3PE
TCF1BV421CD62LBV510GATA3BV421ID2PEcy7
Ki67BV510CTLA4BV421IRF4PERCP cy5.5
IFNgPETbetPERCP cy5.5ID3PE
CD39PECF594EOMEsPE

Tissue collection
Tissue collection
Place mice under deep isoflurane anesthesia (5% isoflurane, minimum of 15 minutes)
Spray the abdomen of the mouse with 70% ethanol, this is to avoid fur contamination of samples
Perform a bilateral thoracotomy, cut the right atrium of the heart.
Collect blood using a 1ml pipette and place in a labelled EDTA tube (2ml K2 coated EDTA)- mix and store at room temperature (RT) until processing.
Dissect the tumor from the skin, place in a labelled 5ml tube containing ice cold RPMI 1640 (no serum); place sample on ice until processing.
Tissue processing
Tissue processing
Blood
Add 1ml RBC lysis buffer (Thermo) directly to the blood tube
Incubate 10min at RT
Centrifuge (500Gx3min) at RT
Remove supernatant and discard
Repeat steps 6-9
Resuspend cells in 1ml PBS;
Transfer to a labelled FACS tube and place at 4oC until downstream processing
Immediately prior to staining with Zombie live/dead dye, centrifuge (500Gx3min), and dump supernatant, retaining the cell pellet
Tumor
Prepare dissociation media, enough for 5ml per tumor (e.g. if 15 tumors prepare 75ml), mix well.
a. HBSS (5ml per tumor)
b. Liberase (5mg/ml): 1:100 dilution (e.g. 750ul in 75ml)
c. DNAse (10mg/ml): 1:500 dilution (e.g. 150ul in 75ml).
Place 5ml of dissociation solution in a 50ml conical for each mouse tumor labelled with corresponding ID
Remove the tumor with forceps and place in a 60mm dish
Use a #10 scalpel to mince the tumor tissue
Transfer the minced tumor tissue into the 50ml conical containing 5ml dissociation media- using the scalpel to dump tumor tissue into the tube
Clean the forceps and scalpel in 70% ethanol.
Repeat steps 16-19 until for all tumors.
Once all samples are minced and in dissociation media, put tubes in a 37C shaker set to rotate at 200rpm for 45min.
After incubation in step 21, use a 5 ml serological pipet to pipet up and down tumor tissue, force through a 70micron cell strainer back into the same 50ml tube, sliding the pipet back and forth over the cell strainer to mechanically dissociate any remaining fragments.
10. Using the same strainer, pipette the solution through the strainer into corresponding FACs tube
11. After all samples are complete, spin at 500gx3min at RT
12. Discard supernatant.
Staining
Staining
Tumor and Blood
Ensure no more than ~50ul volume of cell pellet (using reference FACs tube with 50ul water as guide) is stained, remove cell volume as needed to maintain under this amount; excessive cell density can impair staining and introduce artifacts.
Pulse vortex to disperse pellets
Add 100ul of PBS containing 1:500 Zombie NIR (Biolegend, diluted in 100ul per manufacturer instructions) and 1:500 DNAse I (to prevent clumping).
Pulse vortex
Incubate for 15min at RT
Pulse vortex
Add 1ml of FACs buffer (2% FBS in PBS) to each sample to neutralize the Zombie staining
Spin 500G x 3min, dump and dab
Pulse vortex
10. Add mouse and human FC block at 1ul per sample in FACS buffer at 100ul per tube (make a master mix in FACs buffer for ease, e.g. 100x # samples- total volume, 1ul x #samples for FCX amount- add a few to the # samples to account for dead volume).
11. Pulse vortex after adding the 200ul FCX solution for each tube.
12. Transfer 50ul of each sample to second correspondingly labelled tube (4 panels, thus, for sets of tubes).  Incubating at RT for at least 10min after FCX solution addition.
13. Within each tube panel series, generate pooled isotype control florescence minus one (FMO) controls by taking 5-10ul of each sample and transferring to isotype controls.
14. Resuspend antibody master mixes (see panels spreadsheet, all at 1:100 dilution, note include separate isotype control antibody master mixes) in enough FACs buffer to transfer 50ul per tube (need 500ul total volume per 10 tubes- but add a few samples to account for dead volume). Add 1:500 DNAse I.
15. Add antibodies to each tube (minimum incubation of FCX block is 10min)
16. Vortex
17. Incubate for 20 minutes at RT
18. Vortex again
19. Incubate another 20 minutes at RT
20. During the incubation prepare 1x Fix/Perm buffer, enough for 500ul per tube (Invitrogen FOXP3 Staining kit II, per manufacturer’s instructions)
21. Add 500ul of FACs buffer per tube
22. Vortex
23. Centrifuge at 500G x 3min at RT
24. Resuspend cells in 500ul 1x Fix/perm buffer (step 43)
25. Incubate for 45min- 1 hour
26. Add 1ml 1x Perm buffer (Invitrogen FOXP3 Staining kit II, per manufacturer’s instructions)
27. Centrifuge at 500G x 3min at RT
28. Resuspend cells in 100ul 1x Perm buffer containing 1:100 mouse and human FC block, in addition to panel specific antibodies (at 1:100); along with panel specific isotype controls.
29. Incubate overnight at 4C.
30. Pulse vortex to resuspend cells
31. Wash cells in 500ul 1x Perm buffer
32. Centrifuge at 500G x 3min at RT
33. Dump supernatant, dab tube
34. Pulse vortex to disperse pellet
35. Resuspend cells in 115ul of FACs buffer containing 1:500 DNAse I
36. Vortex
37. Cells are ready for analysis on the Flow Cytometer