Quantifying Cholesterol Accumulation in NPC1 Mutant Cells

For fluorescence studies, CHO M12 cells were grown on coverslips, transfected with Lamp2A-GFP as per manufacturer instructions, and after 48 h, fixed with 4% PFA for 20 min, washed and then stained with filipin (0.05 mg/mL) and mounted in Mowiol as described [39 (link)]. Samples were observed using a Leica TCS SP5 laser scanning confocal microscope equipped with a DMI6000 inverted microscope, blue diode (405 nm), Argon (458/476/488/496/514), diode pumped solid state (561 nm), HeNe (594/633 nm) lasers, and APO 63× oil immersion objective lenses (Leica Microsystems, Wetzlar, Germany). Image analysis was performed with NIH ImageJ software as described below [40 (link)].
The quantification of filipin staining per cell (≥20 cells per group) was performed as described [25 (link)] and served to determine the relative amounts of free cholesterol in enlarged, perinuclear cholesterol-enriched and Lamp2A-positive late endosomal vesicles in NPC1 mutant M12 cells.