I am working on splenocytes derived from healthy murine to investigate the effect of some compounds on interleukins and cytokines. I am using Con A as the mitogen to stimulate the lymphocytes proliferation and cytokines production. I am concerned with investigating the level of TGF beta1 when challenged with the tested compounds in presence of ConA. I have searched the literature for papers similar to the procedures I have to perform but I found nothing. can you help me with protocols to test TGF-beta in ex-vivo? what count of cells should I seed in the 12 well plate? for what duration should the cells be primed with the mitogen? how long should the cells be incubated with the compounds and the mitogen? I have the eBioscience ready-set-go kit for TGF beta1 cat#88-8350.
If you're testing cells ex vivo, you need a much longer timepoint that you would in vivo. You may want to try 48 hrs. For intracellular cytokines, the most common method is to use flow cytometry.
Hi Mohammed,
You can seed at 1x10^6 cells/ml. You will need to do a titration to see what the optimal timing should be- but no less than 24 hrs. I use an ELISA (R&D) kit to quantify TGF-b1. You should do a ratio of active TGF-b1 to total TGF-b1. For Total TGF-b1 you will need to do an acidification as per the kit protocol.
Good luck!
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