Hello,
I'm seeking guidance on how to activate my PBMCs without them dying. I have PBMCs frozen down in liquid nitrogen (primary PBMCs, either human or murine sourced - frozen down after established isolation protocol in 90% FBS + 10% DMSO in Mr Frosty). Upon resurrection, I have good cell yields with a number that matches the vial number (give or take some dead cells, 80% viability). If I seed these cells and leave them, they are perfectly viable and just sit there happily - I know this as I have non-activated controls, as well as expected trypan blue counts after a resting period of 18-24 hours (Media = RPMI-1640 with L-glutamine, 10% heat inactivated FBS, 1% P/S, 10 ng/ml IL-2). However, upon PMA + ionomycin activation (PMA = 50ng/ml, ionomycin = 1ug/ml, for 5 hours) they die - determined by trypan blue count lowering and mostly all cells stained dark blue. I have tried alternate stimulation such as CD3/CD28 beads for 24-48 hours on my human PBMCs, but I still get the same result in the end.
Any ideas as to why my PBMCs are dying, and how I can stop this to conduct intracellular staining and proliferation assays? I'm going to try titrating the PMA/ionomycin, and increase the resting period first. No idea if this is the root cause though!
Thanks!
The problem is almost always the quality of the IL-2. Adding a flat amount in ug/mL isn't a very good idea because the amount of properly folded/post-translationally modified cytokine will vary depending on individual suppliers' expression/purification/shipping methods. See if you can get a batch-specific IU/mg number from your supplier so you actually have an idea of how many funtional units of cytokine you're adding to culture. If the supplier won't provide this, I'd recommend finding one that will.
This is especially important for mouse T cells because with mouse it is actually possible to have too much IL-2 present, so it's important to be able to tune the dosing reasonably precisely.
I'd also recommend bringing in your own aliquot of the CTLL-2 cell line so you can do in-house assays of your IL-2 to make sure it's as potent as you think it is. CTLL-2 proliferation is the standard bioassay in the field, so you'll be able to find tons of assay protocols online.
Final thought on IL-2, I'd strongly suggest making up just enough media + IL-2 fresh at the time of media change/splitting. IL-2 will go off VERY fast if you put it in a big bottle of media and keep it in the fridge. You should break your IL-2 solution into small aliquots (around 20 - 50 uL at 100 IU/uL or something) so that you'll use up most of an aliquot to make up 15 mL of cytokine supplemented media at a time. You can refreeze the aliquots 2 or 3 times only, so keep track of how many times you've thawed an aliquot (just use a marker to tally right on the tube).
Other notes:
-PMA/Iono only triggers the calcium flux pathway that would normally get engaged after TCR signaling. There are other important pathways (MAPK and JAK/STAT pathways) that get turned on after T cell activation that don't get activated by PMA/Iono. Therefore, PMA/Iono T cells will only have partial function, and I wouldn't expect them to be very proliferative. I don't know what kind of experiments you're aiming to do, but I'd recommend focusing on anti-CD3/28 stimulation.
-For your anti-CD3/28 simulation, I would suggest reducing the stim time. T cells tend to get overstimulated very easily and, when that happens, they'll just start dying off. If you're using beads, I would keep stim time to about 18h or so (maybe even less) and then promptly removing the beads.
Dear Rebecca Davies
I briefly wanted to reach out concerning your inquiry. Besides the quality of the IL2 please note that it may be useful to consider to fortify your RPMI medium with additional supplements like non-essential amino acids, pyruvate and beta-mercaptoethanol may improve your cell viability during your T cell culture as per:
Article A role for the histone H2A deubiquitinase MYSM1 in maintenan...
Article Ubiquitin Specific Protease 21 Is Dispensable for Normal Dev...
Article MYSM1-dependent checkpoints in B cell lineage differentiatio...
To 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
I hope that helps.
All the best & good luck with your experiment,
Michael
Dear Rebecca, I assume you are doing it in 96 well plates (round bottom) with at least 0.1*10^6 starting cells. In these conditions, you could use PMA (5-10 ng/ml) and ionomycin (250 ng/ml) for 4-8h. With PMA/ionomycin activation, peripheral blood T cells exhibit optimal immunostaining for IL-4 and IL-5 after 4 to 6 hr, and for IL-2 and IFN-γ after 8 to 10 hr
I think you are killing the cells with too much PMA/Iono.
I hope this helps
Regards
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