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CT26 culture and injection


 
  • Look at the tumor cells under the microscope to make sure you will have enough cells for injection.
  • Remove media and trypsinize the cells with ~2 ml trypsin.  You only need to incubate for about a minute.
  • Bang off the tumor cells and wash the bottom of the flask with 10 ml of media.  Transfer into a 15 ml conical tube.
  • 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.  
  • Spin cells down at 1200 rpm for 5 minutes.
  • Resuspend in 10 ml of HBSS, spin again.
  • Wash at least 2 more times.
  • 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.
  • 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.