I tried with twice as concentrated PMA/IONO, tried also 4h and 5h incubations, tried different aliquots of PMA/IONO (but same concentration of course) but it doesn't work !
any suggesting please ?
Thank you all !
Hi Houda,
I use 50 ng/mL of PMA and 500 ng/mL of iono. I also use 10 µg/mL of BFA to keep cytokines inside the cells during the 4h incubation period, because I analyse the cells through flow cytometry then.
Is it your case?
Hello Inês,
Indeed, I use Brefeldin A and monensin to keep the cytokines inside the cells also during the incubation.
Hello Houda,
I suppose you had already checked with a new batch of reagents? Check also if your reagents are not partially frozen when added to the cell culture (as they are kept in DMSO and very sensitive to lower than expected fridge temperatures). Also, how is the overall cell viability compared to your previous succesfull experiments? If lower than expected, you should also check the possibility of mycoplasm infection...
Hope that these general "real life" tips help!
Hello Marcelo,
I will try with new reagents once I get them next week. I made sure that they were totally thawed before I use them.
for the viability, I always check it when I count my cells and it has always been correct I mean not lower than 95%.
Thank you very much for your tips !
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