Imagexpress 9 ultra confocal plate reader

Live cell imaging of HEK293T cells stably transfected with sfGFP_ABCG2 mutant isoforms was performed using an ImageXpress (IX) Ultra confocal plate reader (Molecular Devices, Wokingham, UK), using a plan-apochromat 40× objective, with an excitation wavelength of 488 nm and an emission bandpass filter of 525/50 nm. Cells were seeded in poly-L-lysine coated, 96-well black-walled, clear-bottom plates (Greiner) at a cell density of 3 × 10⁴ cells/well in DMEM 24 h before imaging. Cells were subsequently washed twice with pre-warmed (37 °C) phenol-red free HBSS (Hank’s Balanced Salt Solution, Sigma, Gillingham, UK) immediately prior to imaging.

Live cell imaging of HEK293T cells stably transfected with sfGFP_ABCG2 mutant isoforms was performed either with an ImageXpress (IX) Ultra confocal plate reader (Molecular Devices), using a plan-apochromat 40× objective, with excitation wavelength of 488 nm and emission bandpass filter of 525/50 nm, or with a LSM710 confocal laser scanning microscope (Zeiss), using a plan-apochromat 63×/1.40 Oil Ph3 DIC M27 objective and 2% argon laser, with excitation wavelength of 488 nm and emission collected at 500–550 nm. For confocal plate reader analysis, cells were seeded in poly-l-lysine-coated, 96-well black-walled, clear-bottom plates (Greiner) at a cell density of 3 × 10⁴ cells/well in DMEM 24 h before imaging. For confocal microscopy, cells were seeded at 2.5 × 10⁵ cells/well in 35 mm glass bottom dishes (MatTek Corp®) 24 h prior to imaging. In both cases, cells were subsequently washed twice with pre-warmed (37°C) phenol-red free HBSS (Hank's Balanced Salt Solution, Sigma–Aldrich) immediately prior to imaging.