Mononuclear spleen suspensions are usually prepared by cutting the spleens into small fragments on a 70 µm cell strainer. Gently, press the spleen tissue with the plunger seal of a 5ml srynge and collect the single cells by passing 5 mL of Ca/Mg free PBS containing 10% (vol/vol) FBS through the cell strainer.

Thank You Prof

Manuel Sanchez-Martin

If you want to culture the cells, the splenocyte isolation process has to be performed using aseptic techniques and sterile media.

1. Isolate the spleen into sterile RPMI/10%FBS/1% pen-strep (use autoclaved scissors)

2.Transfer the spleen into a 70 micron cell strainer fitted on a 50 mL tube, gently cut it into small pieces using pair of scissors (sterile). Add media over to prevent it from drying (important)

3. Then using a 1 mL syringe plunger, gently press the spleen tissue pieces through the strainer, while continuously adding media. This is a very critical step, if you are not gentle, you will damage the cells and they will release DNA, which make cells sticky to each other and loose viability.

4. As you press the cells through the strainer, the remnant of the spleen tissue should become white.

5. Once you collect the cells, add media up to 25-30 mL and spin at 2000 rpm, 10 min, at +40C

6. Discard the supernatant and disaggregate the cell pellet immediately (cells should not be kept in pellet for longer duration as they tend to loose the viability)

7. Re-suspend the cell pellet in 5 mL ACK lysis buffer, and incubate on ice for 5 min to lyse Red Blood Cells (The timing is very critical, if you incubate for longer, the white blood cells will also be lysed, so it has to be exactly 5 min). You can change it to 3 min, if your cell viability is low.

8. Following the incubation, add cold 1XPBS or media upto 40 mL and spin at 2000 rpm, 10min, +40C

9.Discard the supernatant, disaggregate the pellet, re-suspend the pellet in 5 mL of media and mix well using a 5 or 10 mL pipette.

10. Filter the cell suspension through a new 70 micron cell strainer, to remove dead cell clumps and other debris

11. Leave on ice for 10 min and count at 1:10 dilution with Tryphan blue on a Hemocytometer

12. Dead cells will appear in blue and lymphocytes will be round and glowing. If it is a good isolation, you should get 95% viability or higher.

13. For the downstream processes, please dilute cells immediately at a desired concentration (mostly 5 million/mL for T cell assays). Cells should not be kept in higher concentrations as they will loose the viability.

14. Cells can be kept on ice, if you want to use them for staining or for any other further depletions/isolation

15. If you want to use them directly on cell assays, it is strongly recommended that you plate them immediately and keep in a 370C/5%CO2 incubator while getting ready for stimulations.

Good Luck !

Thank you so much Wathsala Wijayalath

You can use this protocol if you want to culture..

Take out the spleen from the mice (infected / Naive). Put the spleen on the sieve. Crush the spleen by using syringe top. Add 10ml of PBBS to the sieve after crushing it nicely and collect everything in 50ml falcon. Centrifuge the cells for 8 minutes at 1200rpm and discard the supernatant.

· To the pellet add 15ml of 1X PBS and mix it properly. Take another 50 ml falcon and add 15ml of Percoll (). Gently add the spleen cells to the falcon with percoll using 10ml pipette by slightly tilting the falcon (be careful and do not mix the cells and percoll).

· Centrifuge it for 10 mins, 1200rpm without brakes.

· Take the falcon out. You will see a buffy layer in the middle. Take it out in another 50ml falcon (carefully without mixing it with the debris). Fill the falcon to the top with 1X PBS.

· Centrifuge it for 10 mins, 1200rpm with brakes.

Discard the supernatant and add 1ml of PBS and do cell count.

Or if you just want to do staining I recommend Manuel's protocol

THANKS PROF PAUL