Hello!
- I would like to test the expression of some intracellular cytokines in primary T lymphocytes by FACS. I need to test the effect of a molecule on their expression. I found a lot of protocols, but they all recommend PMA stimulation before adding BFA for 2-4h, then staining. Now, I don't want to stimulate with PMA, I just want to see if my molecule alone increases cytokine expression. Can someone tell me the best condition to do this, i.e. BFA final concentration, and for how long I need to treat cells with BFA? Today I added it for almost 4 hours, but I saw 5 dots in my positive FACS gate (and I know there should be much more)... Thanks for your help :)
Hello, Nicoletta!
You look at this publication, please:
Article Detection of Intracellular Cytokines by Flow Cytometry
In this article, various protocols for staining T cells for cytokines
Hi,
I usually added Brefeldin A for the last 4h with a total cultivation time of 6h to my cell culture [10µg/ml BFA per well).
If your question is about BFA concentration and time than 10ug/mL concentration & 4-5 Hour incubation time is good (as we do in our setting). Few people add BFA after 1 hour of incubation, but we add BFA along with stimulation only.
Incubation time with your molecule can be obtained by running time gradient incubation for it.
if you are doing Immuno fluorescent labelling ,use PFA for fixation maximum for 10 min and use permeability solution like Tween 20 for 10 min and then go for staining
i dont know which make you are using or would have given the exact incubation timing for staining .
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