When doinf CFSE-proliferation assay with spleen cells from mice I get more then 60% of cell death after 3 days, specially in non-stimulated wells. Same protocol works perfect with PBMCs from humans (20% of cell death). Can anyone help me?
Thanks in advance
The CFSE proliferation assay seems to vary with cell-type. We have used it for myoblasts and find a relatively high but consistent leakage which we have to take into account. Perhaps some subpopulations of spleen cells are spontaneously highly leaky
What concentration of CFSE are you using, and what is in your media?
Medium is DMEM with 20% v/v foetal bovine serum, 2% v/v chick embryo extract, 2% v/v (4mM) L-glutamine (Sigma), and 1% v/v Penicillin/Streptomycin (Sigma)). The experiments were to compare 20% and 5% O2 tensions and involved exposure to 10 µM carboxyfluorescein diacetate, succinimidyl ester (CFSE) which is recommended by the manufacturer for lymphocyte marking.
I have tried with both RPMI and DMEM with 10% FCS, L-Glut and P/S.
I have the same problem whether I do CFSE staining or not, so this is not the explanation of the huge cell dead.
Did you add IL-2 to the medium??
Use 10% NBCS for both RPM I &MEM.with 4 mM of l-glutamine (v/v), 100 U/L of penicillin, 100 U/L of streptomycin,
Jordi: In my experience, that's a fairly normal attrition rate for those cells (unstimulated) in that media. RPMI with 10% serum is recommended (I used heat-inactivated FBS); the antibiotics/antifungals are at your option. Your L-glutamine concentration is about twice what I used (I kept mine at 2 mM). Unstimulated leukocytes don't last long in culture. For stimulation assays/MLR assays, you will NEED a source of IL-2 in the media for optimal stimulation; without it, you may get no clustering or division of cells at all, or at best, very limited clonal expansion.
Thanks Christina. I will try with IL2 to get better viability. In any case I have nice proliferation profiles in the "few" surviving cells when stimulated with CD3.
The sooner you stimulate the cells ex vivo, the better your assay will perform. You should look for grapelike clusters of T cells between 6-12 hours during/after stimulation to determine if your assay is working well--and that does not require CFSE! It might be better/easier to make sure that you have optimized that part of the experiment first before you use experimental mice.
Mouse cells are quite sensitive to oxidative stress .. I woudl recommend adding beta mercaptoethanol to your media, maybe ther are happier then..
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