As per my experience, after stimualtion with PMA/Iono and adding BFA after 1hr. It would be fine if you incubate the cells in CO2 incubator for 72 hours. After that you can go ahead with intracellular staining of FOXp3. 

Many thanks guys. Jens I was thinking to gate them as CD3+CD8-CD25high CD127low? Foxp3high after 5 days of a-CD3/a-CD28 and 5 h of PMA/I.. Why did you mention after 7 days? please let me know what do you think.. Regards to all.

Hi,

The best way to characterize Treg is CD3+CD4+CD25highCD127lowFoxp+. Foxp3 is also an activation marker. So, it should be used in combination with surface markers. It is possible  to detect IL10 in stimulated-Treg by flow cytometry. But take care of the kinetic of IL10 production. It occurs at early time point after activation. Here is what we've done: Freshly isolated Treg were stimulated with PMA (5 ng.mL-1) and ionomycin (1 μg.mL-1) at 37°C for 5 hours. After 2 hours of culture, brefeldin A (5 μg/mL) was added.

I would worry about the % tregs after that long of unpurified culture.  Initially they will be 1-2% of the PBMC, but their growth is highly IL-2 depended and tcell will out grow them quickly.  I would suggest testing for cytokines without the initial CD3/28 stimulation if you do not want to purify for them.  Gael is correct on the markers to test for though the addition of intercellular Helios would be a good addition.  We add the Brefeldin, ionomycin, and PMA at the same time and incubate for 4 hrs in our human treg assay.

for Human Take CD3+CD4+CD25Hi+CD127low+Foxp3 and for mice take CD3+CD4+CD25+Foxp3+ cell as Treg cells and before processing for FACS perform a pilot study (Standardization) for secretory IL-10 through ELISA. means collect the culture supernatant after every 6 hours upto 96 hours and then perform ELISA for IL-10 present in the supernatant. Time at which u will start getting high amount of IL-10 in the supernatant is the time at which IL-10 is getting secreted out of the cell. therefore you need to collect cell just before that time point for FACS (as we measure cytokines inside the cells in FACS study).

Hi Jehan,

I am working on Treg and I also want to phenotype Treg cells from stimulated PBMCs to see some surface markers and intracellular cytokines. What strategy are you using to gate Treg from stimulated PBMCs using flow cytometry?

Thank you guys. Dharmendra I've done a small experiment on freshly isolated PBMCs (but unstimulated) and I could see Tregs phenotyped as CD4+CD25++foxp3+CD127low. You may take in consideration the type of stimulant and if it would affect the expression of the surface markers.

Hi Jehan,

I stimulated PBMCs with aCD3/CD28 for 24 hours and I got a very high expression of CD25. Also, as FoxP3 is transiently upregulated on conventional T cells, I am not getting how to phenotype Treg in such a case? What you did in your case? why you restimulated cells with PMA/I in last 5 hours and why you proceeded for foxp3 staining after 5 days? 

Hi Dharmendra

I think a-CD3/a-CD28 stimulation is not the perfect way to detect Tregs in PBMCs mixture as it alters CD25 and foxp3 expression. I haven't phenotyped Tregs under these conditions that's why I've asked this question at that time. I use the protocol above to detect some cytokines in T cells. the only experiment I did for phenotyping Tregs in PBMCs was under no stimulation conditions. Hope this helps..

Thanks Jehan