- Carfully harvest the tumor fromt he leg of the mouse, try to cut the vein last to avoid a ton of blood contamination.
- Place the tumor in a small culture dish with a wire screen and 5 ml of media.
- Smash the tumor into the screen with the texture end of a 10 mL syringe.
- Filter through both a 100- and 40-micron filter (sequentially) into a 15 ml conic tube.
- Bring the volume up to 15 ml with M media. Wash three times with M. After the first wash, resuspend the TILs in 5 ml media.
- Spin one final time for 3 min at 300 rpm (to resuce clumping andn to get rid of remaining chuncks).
- Remove cells for staining or stimulation assays. Remember to use Fc block when staining.
FACS buffer (wash):
Hypoxia probe: 1:100 (directly labeled FITC)