what is the optimum time to stimulate PBMCs. i have gone through various manuscripts and they have mentioned various time frames ranging from 2-48 hours. i also came across many manuscripts which only took one time poiint reaing i.e., 4 hrs. can somebody help me with this and if u could provide me the refrences
Dear Aasim,
what is your stimulus? what are the activation signals? which reagents?
what is your endpoint, your aim? What will you measure?
You need to specify the above parameters for any helpful suggestion.
Regards
i am stimulating them with my protein@ 10 ug/ml and Con-A @ 5ug/ml, i have to isolate mRNA to perform rt-PCR to measure the cytokine response
The questions raised by Yannis Sotsis are very genuine. However depending upon the dose of ther stimulating agent the time may vary. usually there is a dose dependent stimulation of PBMC's
If you want to get robust and reproducible data, I would suggest to do a time-course assay using a representative endpoint by qPRC and/or Elisa. You can try 2,4,6,8,16 and 24 hours (easy to fit in a normal lab day) and run biological duplicate. This way you'll have all the data on hand to move forward!
The optimum time required may be 6-8 hours, but you have to optimized in a time dependent manner e.g. 6hrs, 8hrs or 10 hrs.....depending upon your molecule of interest. The time may vary for different cytokines ?
if its cytokines you're after these will be produced at different rates, say 4-12 hours.
I would say, you need to consider various issues which include, what cytokine your interested in, as some require longer stimulation, the nature of antigens, since peptide antigens for example will not stimulate at the same rate as particulate antigens.
much as it is mainly agreed 6-8 hrs are optimum, setting up some time course experiments would be the best way to go
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