Mdr assay kit fluorometric

Fexofenadine hydrochloride and emodin were purchased from Tokyo Chemical Industry (Tokyo, Japan). Dimethyl sulfoxide (DMSO), terfenadine, verapamil, Dulbecco’s modified Eagle’s medium (DMEM) with high glucose, MEM non-essential amino acid solution (NEAA) and glutamine were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade acetonitrile and water were purchased from Fisher Scientific Korea (Seoul, Korea). emodin, emodin-8-O-β-d-glucoside, chrysophanol, chrysophanol-8-O-β-d-glucoside, physcion and physcion-8-O-β-d-glucoside isolated from R. acetosa were obtained from the pharmacognosy laboratory of the College of Pharmacy at Gyeongsang National University (Jinju, Korea) [27 (link)]. Fetal bovine serum (FBS), N-2-hydroxyethylpiperazine–N′-2-ethanesulfonic acid (HEPES) and Hanks’ balanced salt solution (HBSS) were purchased from Corning (Manassas, VA, USA). Penicillin–streptomycin, Opti-MEM and 0.25% (w/v) trypsin–EDTA were purchased from Gibco (Carlsbad, CA, USA). Phosphate buffered saline (PBS) was purchased from Welgene (Gyeongsan, Korea). An MDR assay kit (fluorometric) was purchased from Abcam (Cambridge, UK).

A fluorometric MDR assay kit (Abcam) was used to measure P-glycoprotein activity. HCT116, HCT116 DEHP, and HCT116 MEHP cells (1.0 × 10⁴ cells/well) were seeded into 96-well flat, clear-bottom, black-wall microplates and incubated for 24 h. Cells were treated with the P-glycoprotein inhibitor, tariquidar (0, 0.1, 1, 10 or 100 μM) for 24 h. Next, 100 µl MDR dye-loading solution was added to each well and incubated at 37°C for 1 h in the dark. MDR indicator dye fluorescence was detected using a spectrophotometer (U-2800A; Hitachi) at excitation and emission wavelengths of 490 nm and 525 nm, respectively. All experiments were performed in triplicate.