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Look at the tumor cells under the microscope to make sure you will have enough cells for injection.
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Remove media and trypsinize the cells with ~2 ml trypsin. You only need to incubate for about a minute.
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Bang off the tumor cells and wash the bottom of the flask with 10 ml of media. Transfer into a 15 ml conical tube.
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You can remove 10 uL of cells at this point for counting or you can count during one of the washes. Take note of you total volume. Dilute 1:1 with trypan blue.
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Spin cells down at 1200 rpm for 5 minutes.
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Resuspend in 10 ml of HBSS, spin again.
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Wash at least 2 more times.
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Resuspend in HBSS, in a total volume that will give you a cell concentration of 1x105 (for TIL assays) or 0.5x105 (for tumor growth) per 100uL.
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To continue growing tumor cells in culture, transfer 1mL of these cells into a new T75 flask, add 10-15 ml M media.
Inject 100uL of tumor cells subq.