For quantitative measurement of MDR1 and MRP1 activities in viable cells, SOLVO MultiDrugQuantTM Kit applies the Calcein-assay technology patented by SOLVO Biotechnology.
The Calcein-assay utilizes the fluorogenic dye calcein-acetoxymethyl ester (calcein-AM), which is a hydrophobic, non-fluorescent compound that readily penetrates the cell membrane. After entering the living cell, calcein AM is rapidly hydrolyzed by endogenous esterases. As a result of the cleavage, highly fluorescent free acid derivative of the dye is formed, which becomes trapped in the cytoplasm due to its hydrophilic character. Since calcein-AM is an excellent substrate of both MDR1 and MRP1, activity of these efflux transporters results in a lower cellular accumulation of the fluorescent calcein (Figure, Left Panel). Addition of selective inhibitors of MDR1 and MRP1 in excess blocks the dye extrusion activity of the relevant transporter and increases calcein acccumulation in the cells.
Activities of MDR1 and MRP1 transporters are reflected by the difference between the amount of calcein accumulated in the presence or absence of the selective inhibitors. This difference is normalized to the dye uptake measured in the presence of the inhibitor and the results of the test are expressed in MDR activity factor (MAF) values. Thus, the result of the test becomes independent from factors influencing the cellular accumulation of calcein other than the activity of the multidrug transporters.
These variables include the differences in the cellular properties (membrane composition, intracellular esterase activity, cell size, cell surface, etc.); and the methodological differences (e.g. use of different equipment, amplification, and individual variables). Since the influence of these factors is diminished by the simple normalization approach mentioned above, the intra- and inter-laboratory comparison of MAF values is possible. BCRP activity is measured using a similar principle: intracellular accumulation of the fluorescent BCRP-specific reporter substrate is measured in the presence and absence of the selective BCRP-inhibitor (Figure, Right Panel). However, the BCRP-specific reporter substrate is directly fluorescent and does not require cleavage by the intracellular esterases.
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