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Protocol
for Preparation of Splenocytes
- Place the spleen in 3ml of media (i.e. RPMI 1640
with 10% FBS) in a small petri dish (35x10mm)
- Using sterile forceps, place the spleen on a sterile
wire mesh screen (200um bar width and 340um open space)
- Push the spleen through the screen with the plunger
of a 10ml syringe into the petri dish from step 1.
Rinse screen with the 3ml of media. The capsule of
the spleen will be retained by the screen. It is important
to minimize the amount of capsule in the media as
this interferes with subsequent purification, so don't
push too hard!
- Bring the spleen mixture up to 8ml with the same
type of media used in step 1
- Mix the suspension by gently pipetting up and down
- Carefully layer the solution over 3ml of Lympholyte-M
(specific for mouse). Do not allow the layers to mix
since the cells will be retained at the interface
of the two layers
- Centrifuge at 500rcf for 30 minutes
- Remove cells from the white buffer coat layer which
will probably appear to be a slightly diffuse layer
of cells at the interface of the Lymphocyte-M and
media
- Bring the cells to 8ml with the same type of media
used in step 1 and centrifuge 10 minutes at ~180rcf
to wash and pellet the cells
- Discard supernatant
- Resuspend the cell pellet in 10ml of the same type
of media used in step 1
- Make 1ml of a 1:10 dilution of the suspension for
step 10 and count cells using a hemocytometer or cell
counter
- Centrifuge cells from step 10 for 10 minutes at
~180rcf
- Discard supernatant
- Resuspend cells at working concentration (typically
2 x 106 / 100ul)
Note: To properly seed a 96 well plate
for growth, place approximately 2.5 x 105
cells per well.
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