Dear colleagues!
I plan to check cytokine production by my WT and KO Tregs following activation, particularly, IL10 and TGFb (TGFb1 or LAP, exactly) as these are reported to be the major immunoregulatory cytokines. My question is: how to detect TGFb and IL10 from Tregs? I`ve spent some time trying to catch them and always fail to get stable or conclusive results. These cytokines are quite tricky and Tregs are also tricky cells, so I need some advice.
Initially, I planned to use standard intracellular staining protocol - get total CD4 T-cells, then stimulate with PMA/Ionomycin and BfA for 6 hours.
Alternatively, stimulate total T-cells with DynaBeads overnight, then re-stimulate with PMA/Ionomycin and BfA for 4-6 hours and check the cytokines. Results: FoxP3 is lost, no IL10, no TGFb1. However, sometimes I got some TGFb1+ cells, but it was not reproducible.
I tried to activate sorted Tregs for 3 days (plate-bound aCD3/aCD28 + IL2) and stimulate with PMA/Ionomycin and BfA for 6 hours and then again test intracellular cytokine expression. Cells survive and proliferate, but again I can`t get conclusive results. The best I got is just a shift in the fluorescence pattern of TGFb1, but again no IL10+ Tregs.
This protocol yields TNFa and IL2 expression in conventional CD4 T-cells stimulated in parallel, so it actually works.
There is an alternative - ELISA of culture medium, which I haven`t tried yet. It requires to change the culture medium for TGFb1 detection and makes the protocol more complicated.
Is it reasonable to optimize the stimulation conditions for intracellular staining (prolong/shorten aCD3 treatment, prolong PMA+BfA incubation, try some other factors, like IL35) or it`s better to use ELISA directly? Has anybody worked on it?
Hi,
I would suggest that sorted WT and KO T cells activated for Treg differentiation and then collect supernatant for ELISA. In this case, I think you don't need to add PMA/Ionomycin and BfA to treat the cells. It would be more simple and easy compared to your optimization conditions for intracellular staining.
Best
We have successfully measured amount of cytokines such as IL-10 from lymphocyte cultures via ELISA in our lab before. If you are working with a pure culture of Tregs, obtained with magnetic beads or flow cytometry/cell sorting for example, then you can be sure that the cytokines you measure in the supernatant are produced by Tregs. As Yi Yan mentioned, you won't need to stimulate the cells if you're using ELISA, unless you're wanting to measure cytokine production in response to a specific antigen/agent.
I would not go much beyond 6 hours to treating with brefeldin A/PMA/ionomycin as these agents are fairly toxic to the cells.
Got it. Actually, most of protocols measure IL10 and TGFb using ELISA. Some protocols used intracellular cytokine staining, so I thought to start from this, as it it's much easier.
Is it reasonable to sort Tregs, stimulate them in vitro for 2-3 days with aCD3/aCD28 + IL2, and then collect supernatant for ELISA? Should I substitute RPMI with something else?
Maybe try cytometric bead array from the cultured cells. Less cumbersome than Elisa
Some ideas:
- include a Th0 differentiation control, because we have observed that naive CD4 T cells activated in vitro all produce quite a lot of TGFb (both mRNA and protein) so it might not be specific to your iTreg culture. Note that the FCS already contains quite a lot of laten TGFb. so maybe include a culture well with medium but without cells as comparison.
- I have used the multiplex kit from biolegend, which is a 'bead-based elisa' on FACS and it was working and quite sensitive. You have two kits/protocols, one without acidification that givs the amount of active TGFb, the other with activation to get the total, active + latent TGFb - for this step I took the supernatant in the cell culture, that was enough. Restimulation could help, not sure. Restimulation kills Foxp3+ cells preferentially, so I am not surprised if you loose some Foxp3+ cells. The biolegend kit is quite cheaper than luminex or so and you can use any FACS machine.
- for timing, I would wait at least two days after activation, preferentially late points like day 3 or day 5. We change the medium at day 3 to stop TCR activation but I don't know if it makes a difference
- I would be surprised if you see IL10 produced by induced Tregs in vitro. in my hands we don't observe it, only in vivo. However, Th17 in vitro do produce IL10 for instance, so it's not a detection problem.
- For measuring IL10, using a reporter mice you can detect IL10 production much earlier than elisa / multiplex, (still not in iTregs for us) but it depends on the availability of the mice. Intracellular cytokine staining for IL10 kind of works with IL10-PE BD antibody for instance, but detection is a little difficult, you need super strong production, like day 5 or day 7 with PMA/Iono/Bref.
- if you do restimulation and measure cytokine in the supernatant, I would avoid brefeldin as it stops cytokine export
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