Quantification of cellular cholesterol levels using filipin imaging and mass spectrometry after cholesterol enrichment. (A) Cholesterol enrichment quantification by means of Filipin staining. Stable CHO K1 (subpanels a and b) and U2OS (subpanels c and d) cell lines were treated with Cholesterol:Methyl-β-Cyclodextrin (1:10 M ratio) complexes for 1 h in culture conditions. Cells were stained with filipin to visualize cholesterol using confocal microscopy. For each condition, both GFP and filipin fluorescence are shown (subpanels a and c; Scale bar = 6 μm). Confocal images were analyzed using ImageJ with the quantification of cholesterol levels for CHO K1 and U2OS for all conditions shown in subpanel b and d, respectively. The mean intensity values of the filipin signal detected per cell for each condition are shown in brackets, with the mock condition set to 1. Data are shown as mean ± SD (n = 4). The statistical analysis was based on a t test (****P ≤ 0.0001). (B) Cholesterol enrichment in CHO K1 plasma membrane fractions quantified via mass spectrometry. Membrane fractions were validated for membrane enrichment and ER contamination via Western blot (subpanel a). Input and membrane fractions were blotted against a plasma membrane marker (the α-1 subunit of the Na,K-ATPase) and an ER marker (calnexin). For details, see Materials and methods. For lipidic mass spectrometry, ratios of cholesterol to PC were determined for the different conditions indicated with the mock condition set to 1 (subpanel b). Data are shown as mean ± SD (n = 3). The statistical analysis was based on a two-tailed unpaired t test (*P ≤ 0.05). Data distribution was assumed to be normal, but this was not formally tested. Source data are available for this figure: SourceData FS4.