Multidrug Efflux Transporter Ligand Screening Kit

• Cat.No.: Kit-2094
• Product Overview: The MDR1/P-gp Ligand Screening Kit is designed for rapidly screening test compounds for modulation of efflux transporter activity in MDR1-expressing cell lines. The assay uses a lipophilic non-fluorescent P-gp substrate that readily diffuses through the plasma membrane, where it is hydrolyzed to an active fluorophore by cytosolic esterases. The resulting hydrophilic fluorophore is neither membrane permeable nor a substrate for P-gp, hence it remains trapped inside the cell. In MDR1-expressing cell lines, the lipophilic pro-fluorophore is continuously extruded from the cytosol by P-gp, leading to a low intracellular fluorescence. Inhibition of P-gp-mediated efflux by a test compound leads to increased intracellular fluorescence. Specific transporter activity is quantified by comparison with fluorescence accumulated in the presence and absence of a saturating concentration of the included selective P-gp inhibitor. The assay is highly sensitive, has a simple no-wash protocol and is highthroughput adaptable. The kit contains a complete set of reagents sufficient for performing 100 reactions in a 96-well plate format.

Specification

• Size: 100 assays
• Applications: Screening and characterization of drugs and new chemical entities for inhibition of native/recombinant MDR1/P-gp efflux transporter.Identification/characterization of cells and cell lines with a high level of MDR1-mediated efflux (i.e. chemotherapy-resistant).
• Storage: Store kit at -20°C and protect from light. Briefly centrifuge all small vials prior to opening. Open all of the reagents under sterile conditions (e.g. a cell culture hood) only. Read entire protocol before performing the assay procedure.Efflux Assay Buffer: Allow to thaw to room temperature under sterile conditions. Store at 4°C.Fluorogenic P-gp Substrate: Reconstitute with 55 µl anhydrous DMSO and vortex thoroughly to obtain a 400X stock solution. Aliquot the stock solution as desired and store aliquots at -20°C, protected from light. Avoid repeated freeze/thaw cycles.P-gp Inhibitor (Verapamil): Reconstitute with 110 µl anhydrous DMSO and vortex until fully dissolved to obtain a 100X stock solution. Store at -20°C, stable for at least 4 freeze/thaw cycles.