Csu 10 spinning disk confocal unit

Live cell microscopy was performed on a Carl Zeiss 200 m inverted microscope equipped with a Yokogawa CSU-10 spinning disk confocal unit, 100 × objective, 488 and 561 nm excitation lasers, and a Hamamatsu C9100-13 EMCCD detector. Any image panel featuring DAPI staining used fixed cells and was collected on a Nikon TE300 epifluorescence microscope equipped with a Nikon digital sight DS-Qi1MC camera using a 100 × objective and illuminated by LED. DAPI stained cells were prepared by inoculating 1 mL of YPD medium with cells from a yeast colony and incubating overnight. Cells were then sedimented by centrifugation at 3,000 g and resuspended in 100 μL of phosphate-buffered saline containing 4% formaldehyde. Fixation proceeded at room temperature for 15 minutes, after which cells were sedimented, washed with 1 mL tris-buffered saline (TBS), and again resuspended in 100 μL of a TBS solution containing 1% Triton X-100 and 200 ng/mL DAPI and incubated at room temperature for 30 minutes. Cells were again washed in 1 mL TBS before spotting on glass slides and imaging.

BSC-1 cells were seeded in 35mm glass bottom dishes (MatTek, Ashland, MD, USA) that had been coated with 20 μg/mL fibronectin (Sigma-Aldrich). The next day, when the cells were 90–100% confluent, the cells were treated with the indicated drug at the indicated concentration plus 0.05 μM TopFluor Cholesterol (Avanti Polar Lipids, Alabaster, AL), diluted in serum-free growth medium, for 18 h at 37°C in a 5% CO₂ incubator. Following incubation, the cells were gently rinsed once with PBS and cell imaging medium (Live cell imaging solution (Molecular Probes) containing 10% FBS and 4.5 g/L glucose) was added to the dishes. The cells were incubated at 37°C in a 5% CO₂ incubator for 30 min. Images were then taken using a 60X /1.45 numerical aperture (NA) Nikon Plan Apo total internal reflection fluorescence oil immersion objective attached to a Nikon Eclipse TE2000-E microscope equipped with a Yokogawa CSU 10 spinning-disk confocal unit, a 512-by-512 Hamamatsu 9100c-13 EM-BT camera, a motorized stage maintained at 37°C, and a Nikon Perfect Focus system.