Rapid assessment of p-glycoprotein–drug interactions at the blood–brain barrier

We report on a cost- and time-effective fluorescent dye (calcein–acetoxymethylester; calcein–AM)-based assay to screen compounds for p-glycoprotein (p-gp) interactions at the blood–brain barrier. This assay enables the use of freshly isolated or freshly defrosted endothelial cells without prior cell culture. Isolated porcine brain capillary endothelial cells (PBCECs) in suspension were exposed to calcein–AM in absence and presence of substrates and inhibitors of p-gp. Accumulation of intracellular calcein fluorescence was measured using a 96-well plate reader or flow cytometry. P-gp substrates and inhibitors significantly increased intracellular calcein fluorescence in freshly isolated suspended PBCEC and in suspended PBCECs after storage in liquid nitrogen. In both models, intracellular calcein accumulation was similar. Relative concentration-dependent fluorescence profiles of cell suspensions were comparable to those of confluent cell monolayers. Western blot analysis showed highest p-gp expression in freshly isolated cells and cells cultured for 7 d. Importantly, using freshly isolated PBCECs in suspension in combination with fluorescence-activated cell sorting analysis reduced experimental time to 4 hrs vs 7 d with cultured PBCECs monolayers, while retaining and even improving feasibility, reliability, specificity, and sensitivity of the assay. In summary, an improved calcein–AM assay using suspended PBCECs for screening of drug–p-gp interactions is presented . This assay is more cost and time effective and more sensitive than other calcein–AM-based methods and can be performed under nonaseptic working conditions. Therefore, this assay is a useful tool in preclinical high-throughput screening.