Cytokine/chemokine detection in culture supernatant - (Nov/09/2005 )

Im trying to measure different chemokines and Th1/Th2 cytokines in stimulated PBMC cultures. I know its optimal to remove supernatants after 24 hours for some cytokines and after 48 hours for others. However I can only remove supernatants at one time point, so I was wondering could anyone tell me which would be the best time point to do this in order to achieve maximum detection of all cytokines. Thanks

i'd say, you should set up two parallel experiments, same cells same stimulant, take supernatant of one at 24h, the other at 48h 8don't forget to include controls for both timepoints!).

or, if your cells are in enough medium, take of 1/3 of the medium after 24h, and the other at 48h (if using ELISA techinque, 50-100µl per sample and tested cytokine are sufficient), so if you cells are in 1ml medium, take off 330µl (enough for 6 cytokines to be measured) after 24h, and leave the remaining cells in 660µl medium till 48h. you may have to do some maths afterwards, but this could work.

i personally would do it in the way i first mentioned.

mike

Thanks for yer reply, however i don't have enough cells to perform 2 parallel experiments everytime and since im measuring cytokines via luminex which is really expensive its not cost effective for me to measure supernatants at 24 and 48 hours. I think im gonna go with taking supernatants at just 48 hours, do you think i will have missed detection of many cytokines at his time point

Broe,

some cytokines peak early (TNF) and some peak late (IL-10). Which ones are you interested in?

I'm measuring 12 cytokines and chemokines: IL-2, IL-4, IFN-gamma, IL10, IL12, IL-6, IL-8, IP-10, MIP1-a, MIP1-b, RANTES and MCP-1. For the cytokines that peak early - as in at 24 hours, are they still detectable after 48 hours or are cytokines that peak late still detectable at 24 hours?

using a luminex multiplex assay, I was able to detect good levels of the following cytokines 36 hours after stimulation of human PBMCs, though TNFalpha and IL12 (in may case) peak early (as i knew from other experiments)....:

IL1beta, IL-2, IL6, IL8, IL-10, IL-12p70, IL13, IFNgamma, TNFalpha.


mike

Continuing on the same subject, I have purified cell populations I want to test for cytokine production.

In addition to PMA /Ionomycin, I will also be stimulating purified CD4T cells w. plate bound anti-CD3 and soluble anti CD28. Are other cytokines(Like IL-2) necessary for optimal cytokine induction, and also to keep these cells alive?

For PMA/Ionomycin, I will be looking at 3 time points, 24, 48 and 96 hours

Which time points would be recommended for anti-CD3+ 28?