Splenocyte Preparation Protocol

2015-01-13

azim58 - 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

Sensitization media:

o Complete (so FBS) RPMI-10 medium (APPENDIX 2) containing:
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

last seen @ RT next to tissue culture hood

40 uM mesh screen

last seen inbetween hoods in tissue culture room

trypan blue dye

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.

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.

Pour spleen and medium into the well of a 6 well plate for smashing.
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)

Collect the splenocytes by centrifugation at 1200 rpm (not Xg) for 5

min at 4°C.

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.

Add 3 mL/spleen of RBC lysis solution (0.83% NH4CI), mix and incubate

at room
temperature for 5 minutes.

Add 10 mL of complete medium to stop the lysis. Centrifuge as above.
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.

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: Spleen Preparation Protocol.pdf