Splenocyte+Preparation+Protocol


 * Splenocyte Preparation Protocol**

file location: S:\Research\CTL\Protocols\Splenocyte Preparation

__Materials__ ice 50 mL conical tube DMEM medium and/or sensitization medium >> 1 mM sodium pyruvate >> 1× nonessential amino acids plunger from syringe for smashing spleen sterile petri dish RBC (red blood cell) lysis solution with 0.83% NH4Cl 40 uM mesh screen trypan blue dye
 * Sensitization media:
 * Complete (so FBS) RPMI-10 medium (APPENDIX 2) containing:
 * last seen @ RT next to tissue culture hood
 * last seen inbetween hoods in tissue culture room

Note: Splenocytes should be kept on ice as much as possible to prevent non-specific stimulation. Note that 1 spleen yields about 70-100x10^6 cells. 1. Harvest spleens and place in a 50 mL conical tube containing ~ 5 mL of complete DMEM medium. Keep on ice while returning to the lab. 2. Pour spleen and medium into the well of a 6 well plate for smashing. 3. Smash spleen with plunger. Pass smashed spleen solution through 40 um filter. Wash plunger and mesh in the media it was in. May want to use some new media for washing as well (perhaps 0-30 mL total) 4. Collect the splenocytes by centrifugation at 1200 rpm (not Xg) for 5 min at 4°C. 5. Decant the supernatant, removing as much media as possible without removing the cells. Tip the tube to the side to decant the supernatant to not be too near the cells. 6. Add 3 mL/spleen of RBC lysis solution (0.83% NH4CI), mix and incubate at room temperature for 5 minutes. 7. Add 10 mL of complete medium to stop the lysis. Centrifuge as above. 8. Repeat the washes twice with 10 mL each of complete medium and centrifuge as above. --if the peptide pulsed cells aren't ready, resuspend in about 10mL of media and allow to sit in incubator. Once you're ready to use the cells, spin them, then resuspend in 10 mL DMEM. 9. Resuspend the final pellet in 5 mL of complete DMEM medium. ---Resuspend in sensitization media instead of DMEM if performing the killing assay after this protocol. 10. Pass the cell suspension through a 40 um mesh screen if there are large chunks of debris leftover, but only do this just before the cells are needed for counting and the next steps. Store the cells at 37 C until they are ready to be used. The cells could be stored in an upright 25 cm^2 tissue culture flask so that the depth of the liquid is low and there is less pressure on the cells. 11. Count the cells using trypan blue method (1 to 100 dilution with trypan blue is probably appropriate (may want to serially dilute 10 uL cells and 90 uL trypan blue or you could try simply 1 uL into 100 uL). 12. Adjust cells to desired density
 * Suggested concentration: 1*10^7 cells/mL which is the same as (1*10^6 cells/(0.1 mL))

Scan of Protocol from Felicia's Notebook: