Scc 038

Differentiated HepaRG cells were cultured in collagen-coated eight-well chambers (SCC-038, Matsunami Glass Ind., Ltd., Japan) according to the manufacturer’s instructions (160,000 cells per well). After culturing differentiated HepaRG cells for 6 days, fluorescence imaging was performed with 2-Me PeER (1 μM) and Hoechst (1 μM). After the fluorescence imaging, cells were washed with PBS three times, then culture medium was added, and the cells were incubated for 1 day. The next day, the culture medium was removed, cells were washed once with PBS (pH 7.4), and 200 μM DMEM was added. Fluorescence imaging was performed to confirm that 2-Me PeER had been removed from the cells. After that, 2-Me PeER (1 μM) and Hoechst (1 μM) dissolved in DMEM were added, the cells were incubated for 30 min at 37°C, and fluorescence imaging was performed again. Fluorescence images were captured using a Leica Application Suite X (LAS-X) with a TCS SP8 and a 40× objective lens. Light sources were a diode laser (405 nm) and an Ar laser (514 nm).

Differentiated HepaRG cells were cultured in collagen-coated eight-well chambers (SCC-038, Matsunami Glass Ind., Ltd., Japan) according to the manufacturer’s instructions (160,000 cells per well). Fluorescence imaging was performed 6 or 7 days after seeding. Rifampicin (20 μM) [inducer (+)] or 0.1% DMSO [control, inhibitor (+)] was added to the culture medium 3 days before fluorescence imaging, if appropriate. The medium containing rifampicin or 0.1% DMSO was replaced every day before fluorescence imaging. On the day of fluorescence imaging, the culture medium was removed, and the cells were washed with PBS (pH 7.4) and placed in DMEM {200 μl, containing 0.1% DMSO [control, inducer (+)] or ketoconazole (10 μM) [inhibitor (+)]}. After 30-min preincubation at 37°C, 1 μM 2-Me PeER (containing CH₃CN as a cosolvent) and, if necessary, 1 μM Hoechst 33342 (H1399, Thermo Fisher Scientific) (containing 0.1% DMSO as a cosolvent) were added, and incubation was continued for 30 min at 37°C. Fluorescence images were captured using a Leica Application Suite X (LAS-X) with a TCS SP8 and a 40× objective lens. Light sources were a diode laser (405 nm), an Ar laser (514 nm), and a DPSS laser (561 nm).

Cryopreserved primary human hepatocytes (lot #S1412T) were cultured in collagen-coated eight-well chambers (SCC-038, Matsunami Glass Ind., Ltd., Japan) according to the manufacturer’s instructions (200,000 cells per well). Fluorescence imaging was performed 5 days after seeding. Rifampicin (20 μM) [inducer (+)] or 0.1% DMSO [control, inhibitor (+)] was added to the culture medium 2 days before fluorescence imaging. The medium containing rifampicin or 0.1% DMSO was replaced every day before fluorescence imaging. On the day of fluorescence imaging, the culture medium was removed, the cells were washed with DMEM (21063029, Invitrogen Corp., Carlsbad, CA), and a solution of 1 μM 2-Me PeER (0.1% CH₃CN as a cosolvent) and 1 μM Hoechst (0.1% DMSO as a cosolvent) in DMEM {200 μl, containing 0.1% DMSO [control, inducer (+)] or ketoconazole (10 μM) [inhibitor (+)]} was added to each well. After incubation for 30 min at 37°C, fluorescence images were captured using a Leica Application Suite X (LAS-X) with a TCS SP8 and a 40× objective lens. Light sources were a diode laser (405 nm) and an Ar laser (514 nm).