P-Glycoprotein-Activity Measurements in Multidrug Resistant Cell Lines: Single-Cell versus Single-Well Population Fluorescence Methods
Effects of P-glycoprotein blockers on calcein-AM efflux obtained by analyzing single cell fluorescence (FL1) or fluorescence concentration (FL1-FC) with a flow cytometer. P-gp activity was followed with calcein-AM as a fluorescent probe. In each flow cytometry measurement, a sample of 10 000 cells was analyzed. (a) Top panels, left: superimposed all-events histograms of calcein fluorescence distribution (log scale) in control MCF-7/Doxo (solid gray histogram) and MCF-7/Doxo preincubated with the P-gp noncompetitive antagonist PSC-833 (10 μM, open histogram). Right: the amount of fluorescence per cell is expressed as FL1-FC (fluorescent light in channel 1-fluorescence concentration) which is the fluorescent light (FL) divided by the electronic volume (EV) determined by the flow cytometer according to the Coulter Principle. The EV distribution of the sample is given in insert. Bottom panels: the two histograms present the mean fluorescence, FL1 (left) and the cell volume normalized fluorescence, FL1-FC (right) without or with 1 μM or 10 μM of PSC833 or verapamil (VRP) for 10 repeated experiments. (b) The same experiments were carried out in Hs578T/Doxo. Data are presented as mean ± sem with independent assays per data point. *, **, ***.
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