I am working with peripheral blood MDSC and I try to show their p-STAT3 level using with ıntracelluler flow cytometry staining. But there is a specificity problem. Iso and p-STAT3 antibody stained the cells are not efficient. Can you suggest me some protocols?
Dear Digdem,
You should contact Prof. Marc Diederich on my behalf (he is registered on RG).
Best regards
Robert
Dear Digdem,
You gave not enough information. What protocol did you use (if I see your protocol, I can compare it with mine protocol and say what do I do in another way)? What Abs did you use (the same story)?.. I have used p(Tyr705)-Stat3 (D3A7) rabbit mAb-Alexa647 from Cell Signaling (concentration 50 ug/ml). Final dilution that I used for staining was 1:50 (therefore the final concentration was 1 ug/mL). The isotype control (rabbit IgG-Alexa647) was used at the same final concentration (1ug/mL).
Success!
Dear Digdem,
Maybe you don't see a signal because there is no signal. Stat-3 phosphorylation has a fast kinetics. You need a positive control to be sure that your protocol is not working. It will give you a better answer than isotype controls. As positive control I suggest you to activate isolated PBMC with IL-6 at 20ng/ml for 15 minutes. You should see a nice signal on CD4 T cells. Also remember to work fast and minimize cell stress before the fixing step. In my experiments I used Alexa Fluor® 647 Mouse Anti-Stat3 (pY705) Clone 4/P-STAT3 from BD and BD Phosflow™ Perm Buffer III.
Raffaello
Dear Digdem,
We detect pSTAT3 phosphorilation to test for integrity of IL10R. We use a mAb to pSTAT3 from BD labeled with PE. The key in our hands is the permeabilization protocol. We stimulate cells in whole blood for 15 min with 5 or 20 ng/ml IL10. Then perform RBC lysis-fixation (BD lysis solution with fixative) and wash WBCs with PBS. After final wash, resuspend cells with 1 ml ice-cold methanol and incubate for 30 minutes on ice. Wash twice with PBS-BSA, and perform staining with pSTAT3 plus a surface marker for 30 min. Then wash and analyze . In spite of the treatment with methanol (and the common opinion of technical handbooks) in our hands at least CD3 and CD14 reactivity is retained.
Hope it heps!
Francisco
- Badges
- Science method