To lyse the cells, you will need erythrocyte lysis buffer (10mL Tris-HCl 0.17M and 90mL Ammonium chloride 0.16M). This one worked nicely for me, but you have to keep cells for a short time in the lysis buffer 30-45sec, otherwise you may loose your spleenocytes.

The media for spleenocyte culture is Complete RPMI1640.

Do not forget to check their viability before their incubation.

all the best kumal :-)

hi Tanedjeu,

Thaks

I also use the ACT lysis buffer as detailed above.  An alternative (a bit more robust you may lose some which blood cells) is the ACK lysis buffer (protocol in link below).

• Place the freshly isolated spleen in a petri dish with PBS.
• Using frosted glass slides - gently press the spleen between the two slides while using a rotating motion to cause disaggregation.
• Spin down cells and resuspend in lysis buffer (ACT/ACK) for 3-5min.
• Wash thoroughly with PBS, cell count, spin down.
• Resuspend cells at ~1-10million/ml in complete RPMI media

Complete RPMI

• 10% FBS
• HEPES: 5 mM (buffering agent)
• Pen: 50 ug/mL (darn bacteria - antibiotic 1)
• Strep: 50 ug/mL (darn bacteria - antibiotic 2)
• Gent: 50 ug/mL (darn bacteria - antibiotic 3)
• BME: 50 nM (2-mercaptoethanol) Absolutely required for primary cell growth)

http://escore.im.wustl.edu/SubMenu_protocols/protocol8.html

Thanks David