Diethylpyrocarbonate depc treated water

A total of 10 SCoT [47 (link)] and 10 [59 (link)] DAMD primers were used in this study. Polymerase chain reaction amplification was performed in volume of 25 μL consisting of 2.0 μL 100 ng DNA, 2.5 μL of 10 x Buffer (Bioline), 1.5 μL of 50 mM MgCl₂ (Bioline), 2.0 μL of 2.5 mM dNTPs (Bioline), and 0.2 μL 500 U Taq DNA polymerase (Bioline), 1.0 μL of 10 μM each of the SCoT and DAMD primers (Additional file 2: Table S2) and 15.80 μL of 500 ml diethylpyrocarbonate (DEPC)-treated water (Invitrogen Corporation, USA). The PCR cycling profile used for the reaction consisted of an initial step at 95 °C for 5 min., 40 cycles of 94 °C for 30s, 55-65 °C for 35 s, 72 °C for 1 min, and a 10-min final extension at 72 °C. Eight (8) μL of the PCR products were electrophoresed in a 1.5% agarose gel containing 0.5 mg/ml ethidium bromide and photographed on Transilluminator UV light (Fotodyne Incorporated, Analyst Express, USA).

Cryopreserved human hepatocytes from three donors (#1: HMC 514, #2: HFC920, #3: HMC1034), the High Viability Cryohepatocyte Recovery Kit, Hepatocyte Culture Media Kit, Matrigel matrix, and collagen I-coated 24-well plates were obtained from Corning Life Sciences (Woburn, MA, USA). GlutaMAX-I supplement was purchased from Gibco (Grand Island, NY, USA). Cryopreserved HepaRG cells, HepaRG Thaw, Plate, and General Purpose Medium Supplement, and HepaRG Induction Medium Supplement were obtained from Biopredic (Rennes, France). Calcitriol, omeprazole, 6-(4-chlorophenyl)imidazo[2,1-b] [1,3]thiazole-5-carbaldehyde O-3,4-dichlorobenzyl) oxime (CITCO), rifampin, bupropion, and William’s Medium E were purchased from Sigma-Aldrich (St. Louis, MO, USA). Superscript III First-Strand Synthesis SuperMix, TRIzol, and diethyl pyrocarbonate (DEPC)-treated water were obtained from Invitrogen (Carlsbad, CA, USA). Quantitative polymerase chain reaction (qPCR) reagents were purchased from Applied Biosystems (Foster City, CA, USA). Dimethyl sulfoxide (DMSO) was obtained from Merck (Merck-Millipore, Darmstadt, Germany). All other chemicals were of reagent grade or better and were used without further purification.