TITLE: Flow-cytometry-based in vitro assay for assessing T-cell-mediated cytotoxicity against a target cell line (24-well plate, pmel-1 or OT-I T cells, MC38 cell line)
AUTHORS: Bulent Arman Aksoy, Pinar Aksoy, Elinor Gottschalk, Jeff Hammerbacher

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In vitro co-cultures of cytotoxic T cells with their target cells are important assays to asses the functionality of the T cells in a scalable way. These assays rely on co-culturing CD8 T-cells, often times genetically modified to express a specific TCR or CAR, with another type of cell line that can be recognized by T cells. Co-cultures are typically run for 6-24 hours and then the amount of cells that were killed in the co-culture can be assesed through different techniques -- e.g. radioactive Cr or non-radioactive LDH release assays. Here, we outline another alternative to these release assays which relies on flow cytometry to estimate the number of target cells left in the culture after a certain period of time.</div></div>

[STEPS]
?. [Day 0 - Seeding the target cells]
Collect at least 3 million MC38s by trypsinizing them from an on-going culture
?. [Day 0 - Seeding the target cells]
Spin them down at  for  at  and re-suspend them in fresh media at a 250,000 cells per mL concentration
Centrifuge: 200 34
4 °C
?. [Day 0 - Seeding the target cells]
Seed each 24-well-plate well with 500 uL of the cell suspension (i.e. 125,000 MC38 cells per plate)
?. [Day 0 - Seeding the target cells]
Incubate overnight and allow cells to adhere to the plate
?. [Day 1 - Co-culture]
Collect 2 million cytotoxic T cells per sample  (i.e. per well) from an on-going culture
?. [Day 1 - Co-culture]
Spin them down at for  at  and re-suspend them in fresh media at 1 million per mL concentration
Centrifuge: 350 34
4 °C
?. [Day 1 - Co-culture]
Supplement T cells with 200 IU/mL rIL2
?. [Day 1 - Co-culture]
Aspirate the culture media from each of the 24-well-plate wells that contain a sample. Try to aspirate as much as possible but make sure you don't disturb the adherent cells during this process
?. [Day 1 - Co-culture]
Add  of the T cell suspension onto each of the sample wells. Assuming that the cancer cell line doubled overnight, this would result in a 8:1 (2 million:250K) T-cell:MC38 ratio.
2 ml
?. [Day 1 - Co-culture]
Incubate the co-culture for at least 16 hours (overnight) before assaying.
?. [Day 2 - Flow-cytometry-based cytotoxicity assesment]
Prepare and label  eppendorf tubes for each of your samples
2 ml
?. [Day 2 - Flow-cytometry-based cytotoxicity assesment]
Using a P1000, thoroughly pipette up and down each well and transfer everything in the well over to the corresponding  tube
2 ml
?. [Day 2 - Flow-cytometry-based cytotoxicity assesment]
Without letting the well to dry out too much (> 2 minutes), add  of trypsin and incubate at  for  .
200 µl
4 Room temperature
?. [Day 2 - Flow-cytometry-based cytotoxicity assesment]
Stop the trypsinization by adding  of culture media into each well
800 µl
?. [Day 2 - Flow-cytometry-based cytotoxicity assesment]
Spin down the tubes at  for  and aspirate  from the top without distrubing the cell pellet. Then transfer the   of trypsinized cells over to the corresponding tube. Spin down the tubes at  for  and aspirate the supernatant.
Centrifuge: 350 34
1 ml
1 ml
Centrifuge: 350 34
?. [Day 2 - Flow-cytometry-based cytotoxicity assesment]
Re-suspend the cell pellet in  flow buffer and add  from each of the antibodies.
1 ml
5 µl
Make sure to pick two markers that will distinguish one cell from the other. We have found CD3 and CD8 staining, together, can help easily distinguish T cells from the cancer cells. Given that it is not easy to gate cells out only using the FSC/SSC channels, having these extra stains increase the specificity.
?. [Day 2 - Flow-cytometry-based cytotoxicity assesment]
Stain the cells for  at  or  at
4 Room temperature
4 °C
?. [Day 2 - Flow-cytometry-based cytotoxicity assesment]
Spin the cells down at  for , remove the supernatant, and re-suspend them in PBS.
Centrifuge: 350 34
400 µl
?. [Day 2 - Flow-cytometry-based cytotoxicity assesment]
Run  of the sample fully on flow (i.e. do not limit the number of events).
200 µl
Since the cancer cells co-cultured with lots of T cells can easily clog the flow cytometer, having half of the sample as a back-up always helps.
?. [Analysis]
To count the number of MC38s left in the dish,- Using your MC38 samples that were cultured on their own first, define the viable population as your initial gate (G0)- Within your G0, open the CD3/CD8 stain channels and gate the double-negative population (MC38s)
?. [Analysis]
Normalize the positive samples against their corresponding negative controls to estimate the fraction of MC38 that are left in the plate. Substracting this from 100% would give you an estimate of how much cytotoxicity happened for that particular sample.
At 8:1 OTI:MC38 ratio, 16 hours co-culture should yield >70% cytotoxicity when the number of MC38s left in the SIINFEKL-pulsed well is normalized against the unpulsed well.
?. [Day 1 - Co-culture]
For positive controls (samples that are expected to get killed), if the cancer cell line doesn't express or present the target protein/epitope, make sure to supplement the co-culture with the target peptide.
For pmel-1 CD8 cells, the co-culture should be supplemented with the hgp100 at 0.96 ug/mL (i.e.  ) at the beginning of the cultureFor OT-I CD8 cells, the co-culture should be supplemented with the SIINFEKL at 9.63 ug/mL (i.e.   at the beginning of the culture