How long is your stimulation? I have always done 4 hour stimulations. This does not appear to be long enough for the TCR to downregulate while still resulting in high cytokine production. I only noticed significant TCR downregulation when I tried 24 hour stimulations.

I just started intracellular staining in PBMCs and I think PMA is responsible for downregulation of CD4, CD8 and possibly TCR (I havent look at TCR yet). My friend reccomended using shorter time for stimulation. After just 6h of PMA stimulation you should be able to see a response (in my case IFNg production) and still identify T cell markers. I used 24h and wasnt too bad, but with 48h I could not see either CD3, CD4 or CD8 on T cell population. I hope this helps.

You can try using 50ng/ml PMA + 1uM iono for two hours. Then, add BFA 5ug/ml for additional 2 hours.

Dear Maria,

we do see the same phenomenon of CD4 and also CD8 down-regulation after stimulation with PMA/Ionomycin. After 6hrs incubation for cytokine assay, it is visible bot not absolute, you can still see CD4+ and CD8+ T cells, but the MFI goes down.

Brefeldin A should not be tha cause as it is supposed to stop the whole vesicle transport and vesicle relase (a point which would anyways be easy to test).

It is known that activation causes a recycling step of the TCR and associated molecules, which is due to down-stream activation of signaling cascades (e.g. see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3138075/).

I would suggest you to either decrease incubation time or to massively play with your PMA/Ionomycin concentration. Whether any other early acitvation marker like CD69 or LFA-1 is also helpful in your case, you'll have to try out.

Good luck!

Try decreasing the time to 2h, it is sufficient to see cytokine production by gdT cells, and use Monensin (together with PMA/Iono from the start) instead of BFA, Good Luck!

You can also try labeling the gd TCR before the 2-4 hour stimulation. TCR is internalized to prevent further stimulation but generally not degraded over this time frame. The TCR-Ab complex would thence be contained in a recycling endosome. If you must do a 16-24 hour stimulation this may not work however. I would also recommend using monensin as above.

PMA causes the downregulation and you really can't get around the phenomenon - it's the nature of the stimulus. I would suggest changing your stimulus to something like the superantigen staphylococcus enterotoxin B (Sigma, Inc; check with your Institute's Safety Department, as some require registering its use because it is listed as a Bio Agent in some areas) - downregulation of the TCR is minimal and you will get good cytokine expression (though not as polyclonal as PMA/Iono).

I agree with the comments above. A long time ago we also tried to stain for surface markers (CD4) intracellulary following the PMA stimulation, which kept the signal detectable for a longer period of time when compared to surface staining only.

J Interferon Cytokine Res. 2001 Jul;21(7):503-6. (Identification of IFN-gamma-producing CD4+ T cells following PMA stimulation)

Hi Maria. Just a comment on your question. In general, activation will result in apparent reduction in CD4 signal. Cells look 'dim' when fully activated and stained for CD4. But it is not necessarily 'downregulation', seems to be a redistribution of the molecule because the cells do increase in size. When analzyed by flow, they shift to the right on forward scatter. Your best bet is to reduce stimulation time as suggested above, there will be a short time when your cells are already responding to your stimulation but not yet fully activated.

Though I've never specifically tried with gamma/delta Tcells, you could also try adding your fluorochrome-labeled g/d TCR mAbs to the culture during stimulation. This allows binding to occur and sometimes accumulate before downregulation/internalization/masking of the marker. I've been successful doing this for other markers, such as CD40L, CD3 and CD28. There are potential issues/caveats with doing this, but it may be worth a try.

We found adding CD4 at the intracellular staining step was better than surface staining. You could try this. I would also suggest no longer than 3hrs stimulation.

Dear all, thank you very much for your kind and inspiring answers. You all are really helpfull! But... I have more questions for you :))))

What does Monensin exactly? why is it better than Brefeldin A?

What is the difference between using Ionomycin and Ionophore? I am using the second one in my human PBLs, and Verena Schlaphoff suggested the first. Is this may be a problem as well?.

Is there any special treatment for these reagents (PMA, iono, monensin, BFA...) in order to keep its activity? I have also experienced that with freeze/thawn cycles activation goes worse...

Thank you again!

Dear Maria,

you're right, repeated freeze-thaw cycles decrease the activation potential of PMA and Ionomycin, at least in our experience. We have PMA stock in DMSO frozen at -2°C and do strong dilutions with PBS for usage to diminish DMSO effect (it is toxic at high conc. inhibiting proliferation). I don't know exactly the difference between Ionomycin and Ionophore, though Ionomycin also acts on the Ca2+ transport. We also have a stock in DMSO and dilute it with PBS for working aliquots. Again, small aliquots to freeze-thaw each aliquot max 2-3 times. I don't whink that this is your prob, rather the long and strong stimulus. Also BrefA we have in small aliquots at -20 and usually thaw one aliquot max twice.

As to BrefA vs. Monensin: BrefA inhibits retrograde vesicle transport from the Golgi apparatus to the ER. Monensin likewise blocks protein transport, but the mode of action is different through a Na+-dependent mechanism, as far as I know. In some cases (that is for some cytokines) Monensin seems to work better than BrefA. I have no experience in combining the two, but I guess it should work if concentration is not too high (e.g. limit both to half the concentration as to single use).

Keep in mind that both are toxic when used for longer time. In my experience max 12hrs is okay, afterwards your cells die. We usually do 6hrs incubation, one without and 5hrs with BrefA.

Hope this helps.

Cheers

Verena

Thank you very much Verena. I forgot to mention that my incubation time is 4 hours, PMA, iono and BFA all added together to O/N cultured PBLC (thawn the day before). So I thought it was quite mild activation... Probably I am adding too much PMA or... my cells are not in good shape after thawing. I have been advised to stimulate cells with CD3 + IL2 O/N in order to improve cell "shape"/viability prior re-stimulation with PMA, but of course these treatment induces the endocytosis of the TCR/CD3 complex, so I can not phenotype TCRgd either...

I will try the monensin, the intracelullar staining and a 2 hours stimulation.

Thanks again for all the suggestions! :D

Hi Maria

I have the same problem with you, I use PMA at 10 ug/ml and 4 hours activation for PBMC. After all, run on Flow and I couldn't find any CD4+ T cell.

Would you mind sharing the condition that you use and get the good result ? is it work when reduce the incubation time to 2 hr?

Thank you in Advance Maria J. Garcia-Leon

Suvichada A.