spleenocytes die in culture - spleenocytes die in culture when put for invitro stimulation (Aug/01/2002 )

Dear Netters,
I am having trouble getting mouse spleenocytes to survive in culture for more than 3 days. I am trying to standardise CTL assay and am encountering this problem. The viability falls precipitously by the 4th day. The conditions I use are: Isolate spleenocytes, purify by histopaque and put the into culture in RPMI -10%, with or without IL-2. Please give me hints as to how to go about setting up CTL assays.should I be using any more factors in my media? What is the role of B-Mercaptoethanol in the medium? Thank you in advance
Lakshmi

Hi!

I too work with mouse spleenocytes, preperation is basically the same as you do it, except I do Ery-lysis (resuspend cells for 5 minutes in 150mM NH4Cl,1mM KCO3, 0.1 mM Na2EDTA, pH 7.2 - 7.4; then centrifuge for 5 minutes at 150g).

I use RPMI 1640 + 5% inactive FCS, 20mM HEPES, 50µM 2-Mercaptoethanol.

My cells look good for at least 3 days, which is the time I harvest them. So I don't konw really about longer culture, but as said, at 72h my cells look fine.

About that 2-ME thing. I was told - in the fashion of an "urban legend" - that 2-ME somehow "stabilizes the cell membrane" and is therefor utterly important.

Mike

Never give up, never surrender!

Does your medium contain Glutamine? If not, add to 2mM final concentration. It may also help to add pyruvate and Hepes, but in my experience, this is not necessary if mixed suspensions of all cell types (except erythrocytes) are put into culture. b-mercaptoethanol is a standard ingredient for mouse lymphocyte media, but don't ask me what it is supposed to do there.

Another thing: spleen cells like close contact to one another. If using 96-well plates, use the round-bottom type, and put -if possible- at least 100000 cells/well.

I hope this is of some help to you.