I stimulated PBMC with PMA at 50ng/ml , ionomycin at 1ug/ml , and BFA at 5ug/ml for 5 hours, and I also tried to stimulate PBMC using LPS (100ng/ml) for 24 hours, both methods were failed. I found that others suggested to add BFA at the last 2 hours. I want to know why BFA can add at the last 2 hours? If we add BFA more earlier, it seems to can get more IL10?
Hello Liu,
in my opinion, you have to stimulate your cells "overnight" (18 hours) with PMA/IONO and ONLY for the last 6 hours of incubation you have to use the Blocking Mix, composed by BFA, Monensin and DNase.
It's very important use the Blocking Mix for a short time because it is toxic for cells.
I hope it was useful,
Good luck!
Hello Liu,
the blocking mix is needed when you performe an intracellular staining, such as to detect cytokines.
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