Quantitative Analysis of Drug Metabolizing Enzymes in THLE-2 Cells

TeamChips were rinsed twice in PBS for 10 min each, followed by fixation with 3.7% formaldehyde in PBS for 20 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 10 min^{45 (link)}. The chips were rinsed twice in PBS, incubated overnight in a blocking solution (SuperBlock, Pierce), and then rinsed three times in PBS-T for 5 min each. The primary antibodies (anti-CYP2C9, anti-CYP3A4, or anti-UGT1A4 at 1:250 dilution in PBS-T containing 1% (w/v) bovine serum albumin (BSA)) were then added to the TeamChips, and the chips were incubated overnight at 4°C. The chips were washed three times in PBS-T for 15 min each, and the secondary antibodies (HRP-conjugated goat-anti mouse or rabbit IgG, Invitrogen) at 1:500 dilution in PBS-T containing 1% (w/v) BSA were incubated with the TeamChip for 3 h at room temperature. A tyramide signal amplification kit (Invitrogen) was used according to manufacturer’s instructions to detect the human drug metabolizing enzymes expressed in THLE-2 cells through fluorescence analysis. The scanned images were obtained from the GenePix^® scanner and analyzed by GenePix^® Pro 6.0. The fluorescent signal from actin was used as an internal control.