Biochemical pathway evaluation

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P-glycoprotein Inhibition (Calcein-AM assay)
This in vitro assay utilizes the fluorogenic dye Calcein-acetoxymethyl ester (Calcein-AM), which is a P-gp substrate that rapidly penetrates the cell plasma membrane; once inside the cell, the ester bonds are cleaved by endogenous esterases, transforming the Calcein-AM prodrug into the fluorescent Calcein that is not a P-gp substrate. MDR cells expressing high P-gp levels rapidly extrude Calcein-AM from the cell membrane before it can be hydrolyzed by esterases, thus reducing Calcein accumulation. A P-gp inhibitor will impair that mechanism, resulting in an increased fluorescence.