For evaluation of ABCB1 inhibitors, cells were seeded in black 96-well plates suitable for fluorescence measurements (six technical replicates per treatment) and cultured for 24–48 h. Then, cells were treated with RPMI 1640 medium supplemented with 0.5 µg/mL of the fluorescent ABCB1 substrate rhodamine 123 (Rh123; J&K Scientific, Beijing, China) for 15 min alone or in combination with elacridar or tariquidar. After removal of the treatment and repeated washing with PBS, RPMI 1640 medium alone or supplemented with either elacridar or tariquidar was added to the cells. Immediately thereafter, cells were imaged in intervals of 10 min over 3 h using the IncuCyte S3 Live-Cell Analysis System (10× objective, green fluorescence, 1 image/well).
In order to evaluate the influence of the glycosylation status on the ABCB1 efflux activity, cells were seeded in black 96-well plates in medium supplemented with or without tunicamycin (six technical replicates each). After 96 h, cells were treated with RPMI 1640 medium supplemented with 0.5 µg/mL Rh123 for 15 min. After removal of the treatment, repeated washing with PBS and addition of fresh RPMI 1640 medium, cells were imaged in intervals of 10 min over 1 h using the IncuCyte S3 Live-Cell Analysis System as described above.
In order to evaluate the influence of the glycosylation status on the ABCB1 efflux activity, cells were seeded in black 96-well plates in medium supplemented with or without tunicamycin (six technical replicates each). After 96 h, cells were treated with RPMI 1640 medium supplemented with 0.5 µg/mL Rh123 for 15 min. After removal of the treatment, repeated washing with PBS and addition of fresh RPMI 1640 medium, cells were imaged in intervals of 10 min over 1 h using the IncuCyte S3 Live-Cell Analysis System as described above.