Plan apochromat 63 1.40 oil ph3 dic m27 objective

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.

To confirm expression and membrane localisation of protein constructs cells were imaged live using confocal microscopy. Cells were seeded at 2.5 × 10⁵ cells/well in 35 mm glass bottom dishes (MatTek Corp) 24 h prior to imaging. 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 on a LSM710 confocal laser scanning microscope (Carl Zeiss, Jena, Germany), using a Plan-Apochromat 63×/1.40 Oil Ph3 DIC M27 objective. Images (1024 × 1024 pixels with 8-bit image depth) were collected using an argon laser, with excitation wavelength of 488 nm and emission collected either at 500-550 nm (GFP-tagged proteins) or 493-598 nm (SNAP-AF488 labelled proteins) using a 90 μm pinhole. Gain and offset settings were adjusted to maintain signal within the linear range of the detector and were maintained within each experiment.