I would like to identify IL-10 producing macrophages in the BAL of RSV-infected mice. I didn't see any IL-10 at 7 days post-infection following 3hrs in brefeldin A, surface stain, fixation, saponin, then IL-10-APC. I would appreciate any help with: 1) stimulation necessary? if so, PMA/iono? 2) is monensin better than brefeldin A? length of incubation? 3) is saponin the best permeabilization agent? It is hard to know if my protocol is bad or if I just had bad timing (later in infection might be better). Thanks very much!
Sometimes, It is eassy to identify IL-10 production under stimulation with PMA (250 ng/ml) + Ionomycin (1 µg/ml), for 4 h at 37º C
I agree with Isabel and would recommend using PMA/Ionomycin re-stimulation together with Brefeldin A - otherwise it will be very hard to detect IL-10.
In addition you might have a problem with your fixation/permeabilization protocol. Concentration of PFA is criticial for good intracellular cytokine preservation and depends on cell type and intracellular cytokine - for IL10 in macrophages most likely 1% PFA should be fine. However, I would recommend using the BD Fix/Perm kits which in our hands work very nicely.
Thanks so much for your responses! We will do a little troubleshooting and hopefully have better results.
Your SOP appears to be a good one, let me ask however, what kind of positive controls are you using and are they working as expected? I also would try the BD kits
Omar- that was part of our problem. We did not have any positive controls. We did see that you can purchase cells from BD as positive controls. Before we spend money on those we are trying to differentiate them. We didn't have immune complexes and we have a bit of a time crunch so we are differentiating J774's with IL-4, IL-13, and IL-33. Not ideal but theoretically we should see some IL-10.
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