Calcein Assay® / Hoechst Assay
SOLVO patented Calcein Assay® provides information on the interaction between MDR1/P-gp (ABCB1) or MRP1 (ABCC1) transporters and the test compound. Non-fluorescent, hydrophobic calcein AM, dissolved in the lipid bilayer is pumped out of the cell by MDR1/P-gp (ABCB1) or MRP1 (ABCC1), thus keeping the intracellular concentration of calcein AM low. Modulators of the transporter activity reduce the rate of calcein AM efflux, leading to increased intracellular calcein AM, which is then hydrolyzed by intracellular esterases, making it fluorescent. Free calcein is hydrophobic and thus retained inside the cells.
With no inhibitors, the rate of calcein accumulation (and fluorescence) is slow; however, the addition of inhibitors of the transporter results in a faster rate of calcein accumulation. By measuring the reporter compound in the presence of the test article (typically in 8 concentrations), negative control and positive control, IC50 can be obtained, defined as the concentration required to inhibit the transport of the reporter substrate by 50%.
Hoechst Assay is also a whole-cell based dye transport assay that addresses BCRP membrane transporter in a similar fashion to Calcein Assay.
A similar dye transport-based assay, utilizing Hoechst 33342 has been worked out to measure the activity of BCRP (ABCG2/MXR). Hoechst 33342 is a non-fluorescent substrate of both MDR1 and BCRP, and the dye becomes fluorescent after entering the cell and binding to DNA.
Working principle of the Calcein assay
Inhibition of Calcein AM efflux from K563-MDR cells by different concentrations
of test drugs
For a complete list of dye transport assays available as screening services please click here.