How many PBMC per well should I have when I want to stimulate T cell with CD3/CD28 beads?
I will only run them on Flow. In this experiment I want to see the expression of the three surface marker on T cells, how many PBMC should I put in one well? Last time I stimulate 500,000 PBMC per well and then do the flow, the result is very good. But also cost me 12ul of the CD3/CD28 beads.
I want to know usually when you guys stimulate T cells with CD3/CD28 beads. How many PBMC you put in one well? How many cell will have a good result on flow ? and If I set different time to stimulate them like 24 hr and 48 hr, should I keep the PBMC without stimulation stay at the some condition at medium as 24 hr ,48hr ? or thaw a new PBMC from the same donor to stain them as a control?
Hello Yunuo,
i think that the amount of cells/well is depending on the incubation time, staining protocol (e.g. extra- and/or intracellular staining) and the markers that you're looking at. If you look for example at IFNg, you still get a nice signal if you start with 200k cells. I usually stimulate PBMCs ON with CD3/CD28 beads and still get nice responses. Why don't you do a serial dilution to determine the minimum amount of cells...
Rgds
Thank you ,your answer is really helpful! I am planning a serial dilution of the cells today !
It also depends whether you have to divide your sample for different stainings, or that you can combine all activation markers into one staining? And how many control stainings you have. If you can do with one mix of activation markers and one control sample you should have enough with 50-100.000 cells. Also depends if you are interested in CD4 or CD8 cells. Usually you should have >1000 cells in each population that you are looking at to have an accurate measurement. So if you use 50.000 cells, assume CD4+ T cells are ~30-60% of PBMC, you should have a minimum of 15.000 CD4+ cells. Divided by two stainings that leaves you with 7.500 CD4+ cells per sample. Even if you would lose half of the cells in the whole procedure you should still have plenty to analyse.
Jolien, when I stimulate CD8(+) cells with CD3CD28..the expression of cell marker works so good in fresh PBMC but not so good in the frozen Patient PBMC..but I check the paper .they should be the same..can u give me some hint if you know how to solve this situation ?
Which markers are you looking at? And how do you freeze your cells (which medium)?
In my experience, T cell proliferation is also a little bit less in frozen T cells, but you should still see clear differences with stimulation. Do you have a lot of cell death after freezing?
I plate .2 x 10(6) PBMCs per well of TC-96 plate for CD3/CD28 overnight stimulation
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