Two comments, First is that it appears for most cytokines, monensin is the more robust transport inhibitor. A nice comparison can be seen in a number of different Pharmingen publications now circulating. We have noted the same differences in our lab both with human and murine lymphocytes. Point number two deals with use of CD3 and CD28 for stimulation. First, you want to allow the cells to activate without a transport inhibitor (I think 2 to 4 hours is typical). Add the transport inhibitor after the cells have had some time to activate. This gives the cells time to cycle TCR's and prompt a strong response (read Lonzavecchia's Nature paper on serial triggering to understand this necessity). Hope this helps and good luck Keith Bahjat Graduate Assistant University of Florida College of Medicine kbahjat@ufl.edu Qi, Hai wrote: > Some of you may be doing intracellular staining for cytokines. I am > particularly interested in staining mouse lymphocytes under conditions as > close to ex vivo as possible. I am comparing cytokine responses of normal > and infected mice. My experience with PMA and ionomycin plus brefeldin A was > that relatively higher frequencies of cytokine-producing cells could be > obtained, but the difference between normal and infected mice seemed > obscured. I tried Ag and anti-CD28 plus brefeldin A as in vitro activation, > the frequency of positively stained cells was below 1% and such a number was > hard to reproduce. > > Wish to hear from you, and we can discuss more details. Thanks in advance. > > Hai Qi > Dept. of Pathology, UTMB
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