Hi Katy,
This is the method I use to prepare my splenic lymphocytes. It seems to work
really well
as I get about 5x10^7 cells per 5 week old Balb/c spleen. The cells are
usually about
97% viable after this process. The Histopaque-1083 (from Sigma) is specially
made for
mouse lymphocytes.
Good luck,
Julia
1. Remove spleen from mouse and place into RPMI 1640 medium in ice
2. Place spleen in petri dish and mash using the end of a syringe plunger
3. With a 20mL syringe and 18g needle, suction up the cell suspension and
jet over the
petri dish surface a series of times - also pull up the capsule
4. Take an autoclaved piece of nylon mesh (200?m pore size) and “tent” it
into the top of
a centrifuge tube. Pour the cell suspension and debris through the mesh.
5. Wash 2x with RPMI 1640/10% FCS
6. Resuspend the pellet in Ammonium Chloride Red Blood Cell lysing buffer
(approx
5mL/spleen)
7. Incubate 5’ at RT with occasional shaking
8. Add wash medium to fill the tube and spin 10’, 200g
9. Wash pellet again and resuspend in RPMI 1640/10% FCS
10. Precoat centrifuge tubes with FCS for density gradient separation (1mL
in tube, coat
surfaces and then drain)
11. Underlay the cell suspension with Histopaque 1083 (equal volumes)
12. Centrifuge 30’, 18º, 400g (no brake) (TJ-6 approx 1600 rpm)
13. Remove buffy layer and add to 40mL RPMI 1640/10% FCS
14. Centrifuge 10’, 4º, 100rpm (TJ-6)
15. Resuspend in RPMI 1640/10% FCS and count
defaultuser at domain.com wrote:
> I am Katy Tostado and, I would like to know which is the best technique
> to separate lymphocytes from mouse spleen.
> Thank you very much for your attention. I wait your answer.
>> Katia Tostado
> Universidad de Guadalajara, M=E9xico.
