I'm currently trying to evaluate B10 cells on spleen samples, but I was not capable of detecting IL-10 by flow cytometry at all. After isolating splenocytes I performed two protocols:
A) 6 hours stimulation on RPMI-1640 GlutaMax (Gibco)+ Ionomycin 500 ng/mL + PMA 50 ng/mL.
B) Overnight stimulation on RPMI-1640 GlutaMax (Gibco) + Ionomycin 500 ng/mL + PMA 50 ng/mL.
Brefeldin A (10 ug/mL) was added at the final 5 hours of incubation time on both protocols.
I don't know if this is relevant, but I'm stimulating cells on 6-well plate, 3x10^6 cells/3 mL.
Hi Igor,
For detection of IL-10 your protocol of 6 hours seems okay.
Rather than Brefeldin A, Monensin is recommended for IL-10. The stimulation you mention at the end might be relevant depending on which cells (T cells , B cells) you are interested in.
Also, a good anti-mouse antibody is clone: JES5-16E3.
Your stimulation seems fine. Like Fabian suggested, monensin is said to work better for IL-10. I switched to using a mix of BFA and monensin when doing ICS, just so I am not missing out on CN signals. Cells seem to like it better when they are in round bottom tubes compared to flat bottom wells. So you could try plating them in 5 mL polypropylene round bottom tubes. Your cell concentration (10e6/mL) is fine.
Did you look at any other cytokines in your experiment (IFN-g maybe)? If you saw signals for IFN-g and none for IL-10, this means your stimulation/staining is fine and that you are not seeing IL-10 coz it is not there (your cells haven’t expressed any). What staining kit/protocol did you use? If you got signals for other cytokines with your reagents, it also confirms that the problem is not with the reagents you used.
While IFN-g and TNF are expressed at relatively high levels (TNF at 6-12 hours and IFN-g at 6-48 hours), IL-10 is not expressed as much and so chances are you will have trouble picking it up. In my experience, PMA + ionomycin stimulation induces a lot of inflammatory cytokines but not IL-10. If you are interested in detecting IL-10 for experimental reasons, try stimulating your splenocytes with LPS. Then try different time points (6 hours, 24 hours, 48 hours) and pick the one where you see the highest expression.
I found a paper which might be useful. They seem to be using PMA+ionomycin+LPS.
https://link.springer.com/protocol/10.1007%2F978-1-60761-869-0_7
Thanks once again Dra. Joseph! I've read some recent papers and they indeed use LPS along with Ionomycin and PMA. I'm going to add LPS on the protocol that I described in order to check if I'm capable of detecting IL-10.
I didn't try to analyze other cytokines, because IL-10 it's the only interested in. Although it may be a good idea look at other cytokines in order to see if stimulation protocol is actually working properly.
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