Hi everybody!
I´m trying to improve my protocol for CD4 T-cell expansion isolated from murine spleen and lymphnodes.
Do you have any suggestion?
Thank you a lot!
If you can describe your current protocol, we could give you suggestions on how to improve it.
What I´m doing now is just isolate the cells form spleen and lymphnodes and select the CD4+ fraction with magnetic beads.
Regarding the cell culture I´m using RPMI as medium, supplemented with FCS, P/S, ITS and beta-mercaptoethanol. The firts day, the cells are plated in a plate coated with CD3 and CD28; then for each passages I put them the normal 6-wells plates, and I add IL2 and 7 (30U/ml and 0,5ng/ml respectively)
Stefania Pezzana Hey! We usually do this too. RPMI works very good, I also did with Ex vivo medium and it worked pretty well too. How much FBS are you using to supplement your media?
Did you try only with IL-7? Cause IL-2 can also go to Treg cells.
=D
Luísa Lemos No, I didn´t! I´ll try maybe to reduce the concentration. The problem is that I don´t have a "boom of proliferation" (I´m counting the cells with trypan blue).
Maybe there is a problem of concentration...and I was thinking it should be related to the interleukines.
Furthermore, for how long are you able to keep these cells in culture?
The concentration of FCS that I´m using is 10%.
Thank you!! :)
We find that magnetic beads purification is often not a pure enough CD4 population, so we cell sort only the naive CD4 T cell population following magnetic enrichment by inclusion of other surface antigens eg CD3, CD25, CD45rb, CD62L ...
Have you tried using a negative selection... sometimes people report problem with functional assays following positive selection.
Also, for subsequent passages are they kept on CD3/CD28 coated plates? They might be exhausted?
Do everything as you do but add ConA to the culture. It is a great mitogen for mouse T cells
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