Filipin 3

Adherent mNSC cells plated on 35 mm glass bottom dishes (#D35-20-1.5-N; Cellvis) were fixed with 4% PFA for 10 min and washed three times with PBS. Fixed cells were stained with 1 µg/ml Nile Red (#N1142; Invitrogen) or 50 µg/ml Filipin III (#F4767; Sigma-Aldrich). Filipin III was reconstituted with anhydrous DMSO (#276855; Sigma-Aldrich) at 2 mg/ml and used either the same day or within 2 wk (stored immediately after DMSO reconstitution at −80°C). Since Filipin III is very unstable and bleaches rapidly, cells were stained one sample at a time with Filipin III freshly diluted to 50 µg/ml in PBS containing 500 nM nuclear counterstain siR-DNA (#CY-SC007; Cytoskeleton, Inc.) for 45 min in the dark at room temperature. Cells were washed once with 1.5 ml PBS and imaged immediately. Alternatively, fixed cells were stained with Nile Red (#N1142; Thermo Fisher Scientific) for 20 min at 37°C in PBS, nuclei counterstained with 6 μg/ml Hoechst-33342 (#H1399; Thermo Fisher Scientific), washed three times with PBS, and imaged in PBS.