Hello,
I am currently optimizing a naive CD4+T-cell activation protocol based on plate-bound CD3 and CD28 (both 4 µg/ml) in a TC 96-well plate.
I use RPMI (2 mM L-glutamine) + 10% FBS + 1% Pen/Strep + 50 µM 2-Mercaptoethanol as my medium when plating my cells.
The cells are incubated for 24h at 37°C and 5% CO2.
My question is the following: I only get around 50-60% live cells in my activation experiments (checked with FVS780). How do I improve this percentage? I have read that using IL2 may help (Miltenyi's protocol) but all other protocols that I find never mention this.
Thank you in advance and kind regards
Dear Liselotte Mackens
Please consider using RPMI supplemented with glutamine, non-essential amino acids, pyruvate and beta-mercaptoethanol may improve your cell viability during your T cell culture.
You can find the detailed protocols/ culture conditions here:
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To further improve your culture conditions further, please consider your choice of FBS (Hyclone vs.ordinary GIBCO FBS), as per:
https://www.labome.com/method/Fetal-Bovine-Serum.html
All the best & good luck with your experiments,
Michael
It's a bit late to reply,
you might consider using not NUNC-treated plates, on which the anti CD3 and CD8 stick better and activate all cells, so more viability later (in this case, consider testing also lower doses, like 1 microg/mL of the antibodies for coating).
It's not a problem to have ~30% dead cells at activation, but they should amplify later at day 2-3 (first division occurs after ~27 hours). IL2 is a good idea, can not harm. b-mercapto is important to add for murine cells, you already have it. You also supplement glutamine, which is nice, since it is not always stable in old medium. Some people prefer using IMDM, it seems it has more AAs and induce stronger calcium signaling, but not sure this is the reason.
Hope it helps!
the pre-experimental procedures may play a role - use fresh instead of thawed cells - work on your isolation protocols (both for PBMC and CD4+ isolation)
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