I need to run flow to detect the changes on protein expression after the stimulation of endothelial cell line with vesicles. There are two proteins I need to test, one secreted protein and one surface protein. For secreted protein detection, Brefeldin A is recommended. The treatment of endothelial cells (with vesicles) needs to be around 16hrs. In this case, does anyone know the right concentration I should use for Brefeldin A treatment? Will Brefeldin A treatment affect the expression of surface protein if I need to detect two protein at the same time?
I use 5ng/ml of Brefeldin A for my cell staining (for intracellular cytokine staining) as far as I've seen it doesn't affect the expression of surface proteins in Macrophages but it may depend on your cell line.
I normally treat my cells with whatever stimuli I am using then add Brefeldin A at 5ng/ml for 2 hours. Then I harvest the cells and fix them. Bref A is a transport inhibitor so if you want to pick it up your secreted protein it will now be stuck inside the cell. You will have to fix and permibalise your cells before you do flow. Normally at this stage I leave them in the fridge overnight then do my staining first thing in the morning.
Thanks, Samantha. The concentration for Brefeldin A treatment varies a lot (5ng/ml to 10ug/ml were reported in different protocols). I guess it's more related to cell type and treatment time. I need to stimulate endothelial cells for 16hrs. So, I basically add Brefeldin A at the same time. My intitial test indicated that 1ug/ml is still too high for the cell line I am working with. Now, I am trying to test the lower end.
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