TITLE: CAR T cell characterization by flow cytometry
AUTHORS: Andrea R Daniel

[DESCRIPTION]
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.

[STEPS]
SECTION: Tissue collection
1.      Place mice under deep isoflurane anesthesia (5% isoflurane, minimum of 15 minutes)
SECTION: Tissue collection
2.      Spray the abdomen of the mouse with 70% ethanol, this is to avoid fur contamination of samples
SECTION: Tissue collection
3.      Perform a bilateral thoracotomy, cut the right atrium of the heart.
SECTION: Tissue collection
4.      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.
SECTION: Tissue collection
5.      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.
SECTION: Tissue processing
6. Blood
SECTION: Tissue processing
6.1.      Add 1ml RBC lysis buffer (Thermo) directly to the blood tube
SECTION: Tissue processing
6.2.      Incubate 10min at RT
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6.3.      Centrifuge (500Gx3min) at RT
SECTION: Tissue processing
6.4.      Remove supernatant and discard
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6.5.      Repeat steps 6-9
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6.6.      Resuspend cells in 1ml PBS;
SECTION: Tissue processing
6.7.      Transfer to a labelled FACS tube and place at 4oC until downstream processing
SECTION: Tissue processing
6.8.      Immediately prior to staining with Zombie live/dead dye, centrifuge (500Gx3min), and dump supernatant, retaining the cell pellet 
SECTION: Tissue processing
7. 
Tumor




SECTION: Tissue processing
7.5.      Place 5ml of dissociation solution in a 50ml conical for each mouse tumor labelled with corresponding ID 
SECTION: Tissue processing
7.6.      Remove the tumor with forceps and place in a 60mm dish
SECTION: Tissue processing
7.7.      Use a #10 scalpel to mince the tumor tissue
SECTION: Tissue processing
7.8.      Transfer the minced tumor tissue into the 50ml conical containing 5ml dissociation media- using the scalpel to dump tumor tissue into the tube
SECTION: Tissue processing
7.9.      Clean the forceps and scalpel in 70% ethanol.
SECTION: Tissue processing
7.10.      Repeat steps 16-19 until for all tumors. 
SECTION: Tissue processing
7.11.      Once all samples are minced and in dissociation media, put tubes in a 37C shaker set to rotate at 200rpm for 45min.
SECTION: Tissue processing
7.12.      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.
SECTION: Tissue processing
7.13. 10.  Using the same strainer, pipette the solution through the strainer into corresponding FACs tube
SECTION: Tissue processing
7.14. 11.  After all samples are complete, spin at 500gx3min at RT
SECTION: Tissue processing
7.15. 12.  Discard supernatant.
SECTION: Tissue processing
7.2. a.     HBSS (5ml per tumor) 
SECTION: Tissue processing
7.3. b.     Liberase (5mg/ml): 1:100 dilution (e.g. 750ul in 75ml)
SECTION: Tissue processing
7.4. c.     DNAse (10mg/ml): 1:500 dilution (e.g. 150ul in 75ml). 
SECTION: Tissue processing
7.1.      Prepare dissociation media, enough for 5ml per tumor (e.g. if 15 tumors prepare 75ml), mix well.
SECTION: Staining
8. Tumor and Blood
SECTION: Staining
8.1.      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. 
SECTION: Staining
8.2.      Pulse vortex to disperse pellets
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8.3.      Add 100ul of PBS containing 1:500 Zombie NIR (Biolegend, diluted in 100ul per manufacturer instructions) and 1:500 DNAse I (to prevent clumping).
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8.4.      Pulse vortex
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8.5.      Incubate for 15min at RT
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8.6.      Pulse vortex
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8.7.      Add 1ml of FACs buffer (2% FBS in PBS) to each sample to neutralize the Zombie staining 
SECTION: Staining
8.8.      Spin 500G x 3min, dump and dab
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8.9.      Pulse vortex
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8.10. 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).
SECTION: Staining
8.11. 11.  Pulse vortex after adding the 200ul FCX solution for each tube. 
SECTION: Staining
8.12. 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. 
SECTION: Staining
8.13. 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.
SECTION: Staining
8.14. 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. 
SECTION: Staining
8.15. 15.  Add antibodies to each tube (minimum incubation of FCX block is 10min)
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8.16. 16.  Vortex
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8.17. 17.  Incubate for 20 minutes at RT
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8.18. 18.  Vortex again 
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8.19. 19.  Incubate another 20 minutes at RT
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8.20. 20.  During the incubation prepare 1x Fix/Perm buffer, enough for 500ul per tube (Invitrogen FOXP3 Staining kit II, per manufacturer’s instructions)
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8.21. 21.  Add 500ul of FACs buffer per tube
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8.22. 22.  Vortex
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8.23. 23.  Centrifuge at 500G x 3min at RT
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8.24. 24.  Resuspend cells in 500ul 1x Fix/perm buffer (step 43) 
SECTION: Staining
8.25. 25.  Incubate for 45min- 1 hour
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8.26. 26.  Add 1ml 1x Perm buffer (Invitrogen FOXP3 Staining kit II, per manufacturer’s instructions)
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8.27. 27.  Centrifuge at 500G x 3min at RT
SECTION: Staining
8.28. 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. 
SECTION: Staining
8.29. 29.  Incubate overnight at 4C. 
SECTION: Staining
8.30. 30.  Pulse vortex to resuspend cells
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8.31. 31.  Wash cells in 500ul 1x Perm buffer
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8.32. 32.  Centrifuge at 500G x 3min at RT
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8.33. 33.  Dump supernatant, dab tube
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8.34. 34.  Pulse vortex to disperse pellet
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8.35. 35.  Resuspend cells in 115ul of FACs buffer containing 1:500 DNAse I
SECTION: Staining
8.36. 36.  Vortex
SECTION: Staining
8.37. 37.  Cells are ready for analysis on the Flow Cytometer