Hello,
I'm Trying to mark Tregs population in Horses, I adapted a protocol used to mark Tregs in dogs, and It worked at the beginning. But after 7 months I started my experiment and I haven't had the same results.
After the fixation/permeabilization protocol (I used Fix/perm buffer kit from invitrogen), my negative sample appears like a positive one (marking CD4+ and CD25+) and the isotype tube and Treg appears negative.
I don't know what might be happening...
Thought that the fluorescence might be dropping because of the time to perform the protocol, but I evaluated that by passing the samples in the cytometer, and I get good results before the Fix/perm protocol (I get CD4+ and Cd25+ cells).
Does anyone have any suggestion of what is happening?
Hello
This link is useful
Article Equine CD4+ CD25high T cells exhibit regulatory activity by ...
Good Luck
Good evening dear
Look , try to optimize the compensation of your colours.
Then you must stain the 4 ,25 first then use permfix and wash .
Before stain the foxp3
Which stain you used for CD4,5 and which stain you used for foxp3?
u need to optimize step by step.
Hello,
Thanks for your answers! Fatma, in the protocol I use, I stain CD4 and the primary CD25 antibody and incubate at the refrigerator for 30 minutes and then wash with PBS 1x. After that I stain with the secondary antibody for the CD25, followed by incubation (30') and another wash with PBS 1x. And then I analyze it on the cytometer, and I get good results. Then I use fixperm for 30' (at refrigerator) and wash 2 times with the permeabilization buffer 1x, and then I proceed with the foxp3 staining for 30' and one more wash with the permeabilization buffer. After these steps when I analyze the samples I lost the CD4 and CD25 staining. I do the same steps on my negative tube (except the foxp3 staining), and it gives me an awkward result.. my negative tubes seems to be CD4 and CD25 positive. More positive than the tube with the Isotypes and CD4/CD25/FoxP3.
Marwan, CD4-FITC, CD25 PE and FoxP3 APC.
I think the problem in fixation step and the 2 times washing after fixation.
try to read before fixation and after and before wahing and after and try 1* wash snd 2* wash
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