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culture of spleen cell - (Apr/24/2008 )

I decided to isolate and culture spleen cells from mouse.unfortunately i cannot find a detailed protocol.Would anybody who have experience on it be kind enough as to provied me with a detailed and convenient protocol? My boss was poor.
a thousand times thanks

-zamine-

I isolate spleen cells by mushing the spleen up through a Falcon 70um nylon mesh (cell strainer) using the plunger from a 5mL or 10mL syringe. I do a red blood cell lysis and then analyse my splenocytes.

If you wish to culture the splenocytes, I would imagine that you would need a cocktail with growth factors that support B cells survival/proliferation (depending on how long you want to culture the cells for). Those I believe (though I might be wrong) would be IL-7 and maybe Flt-3L. As a base I would use IMDM with FBS.

Others might have better ideas with respect to the specific media, but in the meantime, that's my 2 cents .

-stemcell69-

QUOTE (stemcell69 @ Apr 26 2008, 10:15 PM)
I isolate spleen cells by mushing the spleen up through a Falcon 70um nylon mesh (cell strainer) using the plunger from a 5mL or 10mL syringe. I do a red blood cell lysis and then analyse my splenocytes.

If you wish to culture the splenocytes, I would imagine that you would need a cocktail with growth factors that support B cells survival/proliferation (depending on how long you want to culture the cells for). Those I believe (though I might be wrong) would be IL-7 and maybe Flt-3L. As a base I would use IMDM with FBS.

Others might have better ideas with respect to the specific media, but in the meantime, that's my 2 cents .


Stemcell is right on how to get the splenocytes - but what do you want to do with your cells? Splenic B cells have to be cultured differently to BM B cells (which are not as mature). You don't need Flt3 for mature B cells. I used to culture spleen B cells in RPMI + 2ME, plus other goodies which I can't remember off the top of my head.

-Clare-