Filipin III Staining

Fischer rat thyroid cells were cultured on cover slips coated with collagen I (20 μg/ml) for 2–4 days. Cells were washed with PBS solution and treated with 5 mM MβCD diluted in normal chloride solution for 1 h in the cell culture incubator. Control cells were treated with 5 mM mannitol diluted in normal chloride solution for 1 h in the cell culture incubator. Cells were washed with PBS solution once, fixed in 4% paraformaldehyde for 20 min, then washed three times with PBS solution for 5 min each time. Cells were then stained for 1 h with 10 μl/ml filipin III (Sigma, SAE0087, 10 mg/ml in DMSO) diluted in PBS in the dark. Cells were washed with PBS two times for 5 min each time, then mounted with Vecta Shield without DAPI. Images were taken (at 20 X) using a fluorescence microscope with a UV filter (Integrated Cellular Imaging Core, Emory University). The filipin intensity was calculated with ImageJ.