Mrp1 fitc

Resistance Protein 1 (MRP1), Lung Resistance Protein (LRP) and P21 Expression In order to evaluate if hAMPE induced PGP, MRP1, LRP and P21 expression alteration, 1x10 6 cells were incubated with 1 μg/μL of hAMPE for 72 h. To evaluate PGP and MRP1 expression, cells were collected, centrifuged (1300×g for 5 min) and incubated with 3 μL of PGP-fluorescein isothiocyanate (FITC -BD Pharmingen, 557,002) or 3 μL of MRP1-FITC (BD Pharmingen, 557,593) for 15 min in dark. After, cells were centrifuged (1300×g for 5 min) and ressuspended in PBS. Fluorescence was determined at 485/20 nm emission and 528/20 nm emission. In order to evaluate LRP and P21 expression, cells were collected, centrifuged (1300×g for 5 min) and incubated with 100 μL of fix solution (IntraCell, Immunostep) for 15 min. After centrifugation (1300×g for 5 min), cells were then incubated for 15 min with 100 μL of permeabilization solution (IntraCell, Immunostep) and LRP (1:50 -Santa Cruz Biotechnology, sc-23,916) or P21 (1:50 -Santa Cruz Biotechnology, sc-6246). After centrifugation (1300 g for 5 min), cells were then incubated with antimouse-PE secondary antibody (1:100 -Santa Cruz Biotechnology, sc-3738) for 20 min in dark. Cells were then centrifuged (1300×g for 5 min), ressuspended in PBS and fluorescence was determined at 485/20 nm emission and 590/ 35 nm emission [30, 31] .