I am fixing HCT116 cells to use with a photo activation probe for cell membrane labeling. I have used 1% and 4% PFA in water as a fixative and fix on ice for 20 minutes. I then quench the reactive PFA groups with 1M pH 8 tris for 1 minute, followed by several PBS washes. When I add my probe to the fixed cells, the probe will aggregate inside cells that have a permeabilized membrane causing a high background signal in some cells and no background signal in other. The cells present as if some are alive and some are dead. I need to attain a uniform background signal and therefore need uniform fixed cell conditions. Does anyone know how to fix the HCT116 cells and maintain either an intact or permeabilized membrane across the whole population?
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