Any advice on the optimal concentrations of PMA and Ionomycin to use in cell culture for thawed human PBMCs which are being stimulated for 4 hours for a multicolour T cell flow experiment. Stimulating in a 96 well plate, 1 million cells in 100ul. Literature is quite variable. Any recommendations would be greatly appreciated.
I want to use PMA and ionomycin stimulate T cell, how much brefeldin A should I add? ?
Hi Suzy
To stimulate 1 million PBMC use 50 ng of PMA and 1 ug of Ionomycin for at least 5 hours in the presence of monensin or brefeldin A. You could evaluate different concentrations of PMA and maintain 1 ug of Ionomycin.
I stimulated B cells with 50 ng/ml PMA and 1 micromolar ionomycin. I think this might be a good starting point for optimization for your assays as well.
I collaborated with a group that whacked thawed human PBMCs with 500 ng of PMA and 1 ug of Ionomycin for 2 or 3 hours; I was amazed when it really worked! for short incubation periods it does not kill the cells and it gives a very good signal to noise ratio.
We work with Jurkat cells (human t-lymphocyte cell line) and stimulate them usually with 10 ng/ml PMA and 2.5 µM Ionomycin for reporter gene assays (24 h stimulation). We have also used the same concentrations for shorter incubation (2-4 h) in case of qRT-PCR with no problems regarding induction etc.
Hi Suzi,
Sorry to add question here instead of answering urs. What is the aim of using PMA/ionomcyine. Is only for brief activation of T cells? Does that affect cell surface markers? or is it mainly to induce cytokines production? I need to do this for my PBLs, but still not sure why?
Thanks
Hi Fatma,
I am using PMA/I as a positive control for cytokine production in my T cell experiments. Stimulation is usually short anywhere between 4-6 hours and can affect cell surface markers e.g CD4 is down regulated. I would recommend you do a literature review on how this stimulation affects your markers. Cytokine production is also effected by the type of protein inhibitor you use i.e. monensin vs brefeldin A. I attached a paper you may find of interest.
Dear Suzi,
I'm a little late, but I think you may help me with a question if PMA/I affects CD4 expression, what did you do optimize your CD4 expression? I'll stimulate my cells with this combo for intracellular staining, but I'm worried about my flow cytometry.
Thank you!
I want to use PMA and ionomycin stimulate T cell, how much brefeldin A should I add? ?
how much brefeldin A should i use for stimulate million jurkat cells with 1-2 microgram/ml calcium ionophore and 10-20 ng/ml PMA?
Hi,
Can anyone please suggest me what should be the optimum concentration of PMA & ionomycin to activate raji b cells and for how much time. I am interested in cd69 activation marker.
Thanks in advance.
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