I am looking to stimulate PBMCs in vitro and to look at intracellular cytokine expression of IL-10 and TGF-b in T cell subsets. I have a couple of questions:
1. Most protocols I have seen use PMA/ionomycin (short stim). I was looking to use CD3/CD28+IL-2 (12-24 hours stim) instead. Is this strong enough and ideal to detect IL-10 and TGF-b?
2. What is the recommended starting number of PBMCs that is required for stimulation in a 24 well plate?
Thanks in advance.
Hi
You can stimulate for 2-4 days depends how good cell lines you are getting. i have read people stimulating for even 4 or more days. Since the procedure is totally intracellular and you have to stimulate the factors, it thus becomes one of the most important detection method for IL10 and TGFb. you can take initial concentration of 106 per well.
with regards.
IL-10 and TGFbeta, wow tough nuts to crack! Take a look at our Current Protocols in Immunology chapter for detailed methods, Detection of Intracellular Cytokines by Flow Cytometry Yuzhi Yin, Alyssa Mitson-Salazar, and Calman Prussin.
IL-10 is a tough nut to crack, typically with frequencies of 0.05-0.3% of CD4 cells after PMA/iono activation. When we were doing this more regularly, 16 hour overnight cultures gave best results. I have no idea what the 4 day figure above is based on. Use a very bright fluor.
Wish I had more things nice to say about ICCS of TGFb. My lab burned through months of postdoc/technician time in the aughts, trying to get that to work. I would do an exhaustive literature search to see what people have done since. TGF-b frequencies are even lower!
For all of this you will need a good positive and negative control, or you will end up chasing your tail. ALWAYS titer your mAbs, particularly the anti-cytokines mAbs. In my experience, homemade PFA & saponin buffers have superior signal to noise relative to the various fix and perm kits.
Have fun Mildred! You can email me at siprussin (AT)gmail if you have more questions or need the reference.
Calman
https://www.ncbi.nlm.nih.gov/pubmed/26237012
Dear colleagues, I have encountered with a problem: we are trying to stimulate PBMC with PMA+ionomycin with the following intracellular detection of IL-17 and IFN-g, but the cells are obtained during the first days after myocardial infarction and after 5 hours of stimulation PBMC are clumping and dying. The conditions that worked fine for us before failed to work now. May be the reason is therapy patients are recieving. I tried to decrease PMA concentration twice (from 50ng/ml to 25 ng/ml) but without any effect. Can you give any suggestions what else is it possible to do?
PMA and Ionomycin are rough on cells on the verge to die (over-activated). You can try a more gentle activation with anti-CD3/CD28. If in whole PBMC, only anti-CD3 should work. If APCs are not there you should use beads with anti-CD28/CD3 or pre coated plate with the antibodies. You can monitor the time, as it's more gentle they might take longer to accumulated the amounts of cytokines you need to show differences.
Quick re-question: are all cells dying or only the activated? If everything is dying, you might have changed batch of ficoll, or any other reagent that can have an impact. I'm asking this because you mention they worked before...
Hello, Rafael! Thank you for your answer - unfortunately I don't have anti-CD3/CD 28 now. Only PHA or LPS - but they did not work for IFN-g/IL-17 stimulation. Only stimulated cells are dying, intact cells are fine. I tried the method in summer in preliminary work, but PBMC were from the random individual, and all workd excellent. Seems that cells after MI with medication are quite different compared to random PBMC, and are much more sensitive to stimulation. But I didn't find the way to please them yet...
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