I am looking to stimulate negatively isolated CD4+T cells in vitro and the main cytokines of interest are IL-10 and TGF-b.
My questions are:
1. What should be my starting count (of CD4+ T cells) per well if I was to use a 96 well plate?
2. At what point during the stimulation should I add brefeldin? There seems to be a bit of variation in published papers.
thanks in advance.
Hi Mildred,
When you say starting count, how and how long for are you planning on stimulating your cells before you do ICS? Are you going to TCR stim them (e.g. with anti CD3/CD28 beads) and keep them in culture to allow for proliferation or do you just do short-term stims that won't change cell numbers? This is important to know since cells shouldn't get too dense. Also, what T cell subsets are you interested in? For a broad idea just looking, for instance, at memory versus naive CD4s, you can get away with a total of 50,000 cells/well. However, if you want to separate further to look at e.g. Tregs or other subsets of lower frequency, I would suggest a minimum of 100,000 cells/well, better is 200,000 cells. In my experience, cell numbers should not exceed 300,000-400,000 cells/well. We usually culture the cells in 96-well round bottom plates and then transfer them to 96-well V-bottom plates for staining since you get a nicer cell pellet after centrifugation.
I hope this is helpful, good luck with your experiments!
Sorry, I forgot to answer your question about brefeldin...you are right, there are many different protocols around, every lab seems to have its own approach. I usually restim the cells with PMA/Ionomycin for an hour before adding brefeldin for another 3 hours.
Thank you Christian and yes the above are helpful. I am interested in T regs and I plan to do a short stimulation of isolated CD4+Tcells for 12 hours and then look at specific cytokines in the CD4+CD25+Foxp3+ population. Will this impact on the timing for adding brefeldin that you suggested?
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