Dear Junfen Zhanh,

We used to perform Il17A staining for Th17 cells in our lab. for that we used to stimulate PBMCs with 50ng/mL of PMA and 500ng/mL ionomycin (Brefeldin 10ng/mL after 1 hour in culture) followed by incubation at 37 degree C, 5% CO2 and 95% humidity for 5 hours. After incubation cells should be washed and surface staining is to be done before fixation and permeablization by Cytofix/cytoperm. Alternatively you may use PFA and Tween 20 for perm and Fix.

you should use intracellular staining at the stage of perm/fix. and then you should wash the tube and acquire the tube.

Hope this will help.

Gulab Fatima Rani Thank you so much for your detailed answers.

In my experiment, I analyzed the intracellular level of IL-17 in human PBMC stimulated with PHA at a concentration of 5ug/ml for 48h. We dont have brefeldin A in the lab, and I think it is one of factors.

The T cells are isolated from mice? I know the percentage of IL-17+CD4+ is low, but it is strange that the expression of isotype is much higher than that of IL-17. For this, I have not found an explanation yet.

Gulab Fatima Rani I used the recomended isotype from the same company following the recomended concentration. I think that to try to use a different isotype of same clone is a good choice to exclude the problem of isotype.

Also, the fixation buffer was made from 37% formalin that includes methanol, which may have effect on the intracellular staining. I will add the brefeldinA to try. Hope to get a good result.

Thank u so much!

Junfen

I put the supplement. Today I did the intracellular staining again.

Cell fixation: 200ul of 2% PFA(37% formalin diluted in PBS) for 1x10^6 cells at RT for 15min

Cell permeabilization: 150ul of 0.5% saponin-PBS with 5%FBS at RT for 15min

I think the process of cell fixation and permeabilization was successful, but also got the similar result--isotype higher than that of IL-17A in CD4+T cells. But the level of IL-17A was higher if compared with CD4+T cells without any other intracellular staining but also with the fixation and permeabilization process.

I used the anti-IL-17A-APC(ebioscience 17-7179-42) and the recommended isotype (ebioscience 17-4714-42). Does anyone use the same antibody for the intracellular staining of cytokines in PBMC? Could you give me some advice?

Thank you so much!

Gulab Fatima Rani Thank you so much!

Exactly! My collegue use the same protocol of intracellular staining in PBMC but for other antibody(not cytokines). It is good for his antibody. I think maybe I need to try other clone? It is too complicated.

Hi Gulab,

For intracellular detection of cytokines, culture the PBMC overnight then add mitogen like PHA ( Same conc that you are using) or 5 μl of 10 ng/μl phorbol 12-myristate 13-acetate (PMA) and 1 μl of 1 mg/ml ionomycin (Sigma, Saint Louis, MO, USA) at 37°C and under 5% CO2 for 2 hrs. Add Brefeldin A (10 mg/ml; Sigma) additional 1 hr and then process the sample as per your protocol.

Thanks Mohd. Kamran .

Could you tell me what the is density of PBMC when you culture your PBMC? I seed PBMC at a density of 1x10^6 cells/well in 6-well plate. But I found the cell viability in unstained cells in FACS was too low, about 55%. I read some articles saying that cell concenration of PBMC in the culture would affect T cell survival.

For intracellular staining of cytokine, because I did not use the brefeldin A and used the cytofix/cytoperm buffer made by myself. I thought this was the main reason. I could explain the low expression of IL-17, but it's strange that in all my experiment, isotype always had higher expression than that of cytokines. I tried different intracellular staining protocol and also used different concentration of isotype and IL-17. But it did not work. Maybe do you have any experience for this?

Thank you.

Hi Junfen,

I am using PBMC at a density of 10^5 to 10^6 cells/well but i did not face any problem in terms of viability.

Regarding your intracellular cytokine expression you have to use Golgi stopper otherwise you wont get any cytokine expression in your experiment. If you do not have Brefeldin A in your lab then collect the soup and do the ELISA for your cytokines.

Regarding your viability issue I think there is problem with cytofix/cytoperm .

For permeabilisation you should use saponin and optimise its conc from .05 to 1% and for fixation always use fresh paraformeldehyde.

Also optimise the timing for addition of cytofix/cytoperm from 10 to 20 minutes.

Hope this might help you.

Thank you.

And Thanks Gulaab . Sorry about that I thought it was posted by you.

Hi Mohd,

Thank you for your details.

For the high expression of isotype control, I think it is the problem of antibody. Because I ever tried the cytofix/cytoperm buffer kit, it also showed high expression. Even when I added the BFA, its expression is much higher than that of IL-17.

Which type of PBMC do you use? Human or mice? Frozen or fresh? And normally what is the percentage of IL-17+CD4+T cells in your experiment? I am sorry that I have so many questions at one time.

Thank you so much!