Hello everyone,
I would like to perform an intracellular flow cytometry staining (without extracellular epitopes) to determine cytokine production in BM mesenchymal cells.
It is hard for me to complete the whole procedure on the same day, therefore I thought about splitting the assay and to do it like this:
1. Incubation with Brefeldin A (4-6h, 37C)
2. Fixation with 2% PFA, 10min in R.T
3. Washing with PBS and overnight incubation at 4C
On the next day:
4. Permeabilization with 0.2% Saponin
5. Intracellular staining
I never permeabilized fixed cells after overnight incubation. Is it going to be problematic? Any other suggestions?
Thank you in advance!
Hi Adi,
If you can avoid aggregation during spinning down to collect cells, they will be OK to be sorted. Add 2mg BSA/ml buffer to avoid that in your cell washings in whole procedure.
Regards
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