Dear colleague,
I am doing MC3T3-E1 differentiation. Previously I grow the cell in growth medium (I used DMEM + 10% FBS + 1% PS). When they reach ~ 70% confluency, I changed the medium to differentiation medium (GM + 50 µg/ml ascorbic acid, 10 mM beta glycerophosphate, 100 nM dexamethasone), I also treated some groups with my test material (50 µg/ml). After 7 days, I did ALP staining with BCIP-NBT solution (Sigma; B1911). Before that, I aspirate the medium, wash the cell with PBS, fix the monolayer with 4% paraformaldehyde for 10 minutes, aspirate PBS again, and applied the staining solution for hours (1-3 h). However, I see no color change in the medium (only slightly change under microscope; pic attached). Please kindly tell me if I did a mistake in this procedure. Should I reduce the fixation time, should I increase the cell confluency before inducing differentiation?
Best regards
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