Filipin staining and fluorescence microscopy

Filipin III (Santa Cruz Biotechnology) was dissolved in DMSO at 5 mg/mL, aliquoted and stored under N₂ at − 80 °C until use. CU428 and II8G-IA cultures at ~ 3 × 10⁵ cells/mL were treated with cholesterol in the presence or absence of U18666A as indicated above. After 1 h of incubation, samples of 200 µL were transferred to microcentrifuge tubes, centrifuged at 1000 × g, washed with 1 mL of 10 mM Tris–HCl pH 7.5 and resuspended in 100 µL Tris–HCl. Cells were fixed through the addition of an equal volume of 4% paraformaldehyde—3.4% sucrose in phosphate buffer saline (PBS) for 15 min at room temperature. They were then washed twice with 400 µL PBS and resuspended in 100 µL PBS. Staining was performed with 50 µg/mL Filipin for 1 h at room temperature with rotation in a HulaMixer (Invitrogen). Finally, the cells were washed twice with 400 µL PBS and resuspended in 40 µL PBS.

Cells mounted under coverslips were observed and photographed using a Nikon Eclipse E-800 epifluorescence microscope equipped with an Andor Clara DR-1306 monochrome camera. Filipin staining was visualized using a UV-2A filter cube. After being taken, the images were processed and analyzed using the Fiji software⁴⁴.