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Isolation of TILs

 
  • 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):

1xPBS

10%FBS

1%Azide

 

Antibody concentrations:

Hypoxia probe: 1:100 (directly labeled FITC)