Differences in kinetics of cytokine production between cell types is a limitation of the cytokine flow technique (for example: basophils optimally express I/C cytokines at 2 h, whereas T cells are optimal at 6 h). If you are comparing 2 different cell types will have to pick a time point that is reasonable for both. Ultimately, there is no way for you can answer this question without doing the time course yourself. The kinetics of cytokine expression after the addition of the top-secret activator may be different than that for say, ionomycin. To give you an idea, most people using these techniques for T cell cytokines use 4-6 h for PMA/ionomycin and 6 h for Ag activation. Better to do a time course now, than to do a bunch of work and based on incorrect assumptions about someone's else's kinetics. You never specify when you are adding BFA, although you imply that it is at the end of the experiment. P.S. many monocyte activators, such as endotoxin, down regulate CD14, making discrimination of monocytes difficult. With activation, monocytes may stick to your plastic, making them unavailable for flow cytometric analysis. P.P.S. Please at some point use unlabeled anti-cytokine mAb blocking (Current Protocols in Immunology, Unit 6.24) to verify that your staining is specific. Fixed cells are "stickier" than fresh cells and isotype matched controls (especially in novice hands) do not provide a rigorous control for specificity of staining. Once you have the system working reproducibly isotype matched controls are fine, but in the beginning and for critical or hard to interpret experiments, a blocking control is better and may help to convince skeptical reviewers. > _______________________ > Calman Prussin > Laboratory of Allergic Diseases > NIAID/ National Institutes of Health > > ---------- > From: Maciej Simm > Sent: Monday, August 16, 1999 15:19 > To: Cytometry Mailing List > Subject: cytokine release timeframe > > > Hi again, > > Today's question: We're running an intracellular cytokine staining > for lymphocytes and monocytes in parallel. So we start with whole > blood, add our top-secret :) activators, and let that sit overnight. > Then we plan to add BFA to "plug the hole" and capture any cytokines > the cells are still making. The problem is, for lymphocytes this > time frame is prob. ok (activation from 5pm to 9 am next morning) > b/c it's the amount of time they need to gear up. But are Monocytes > more rapid in their response? I'm afraid that if we do it this way > by the time we add BFA our cells will not be making any more cytokines. > Has anyone had the experience of overnight activation for purpose > of intracellular staining the next morning? > > Any thoughts will be appreciated, > > Maciej S. Simm + Lab > > > === > `---------------------------------------------` > | Maciej S. Simm | 525 E 68th Street | > | Research Technician | Room N-805 | > | Cornell Medical Center | Tel. 212.746.3952 | > `---------------------------------------------` > _________________________________________________________ > Do You Yahoo!? > Get your free @yahoo.com address at http://mail.yahoo.com >
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