Cholesteryl bodipy fl c12

Cells were seeded at 1 × 10⁵ cells/well in a six-well plate 48 h prior to cholesterol extraction. Cells were washed briefly with cold PBS (twice), followed by addition of 1 mL of hexane:isopropanol (3:2) to the wells for lipid extraction, followed by incubation at room temperature for 30 min. The lipid-containing mixture was recovered in an Eppendorf tube and air-dried using an Iwaki Halogen Vacuum Concentrator (IVC-500) for 20 min at room temperature. The pellet was then resuspended and cholesterol content was determined using the Amplex Red^TM Cholesterol Assay Kit (Invitrogen). For reloading cholesterol, cholesterol-methyl-β-cyclodextrin (C4951, Sigma) was added to the culture medium at the final concentration of 10 μg/mL for 24 h^{47 (link),48 (link)}. Incorporation of cholesterol into cells was estimated by adding CholEsteryl BODIPY FL C₁₂ (Thermo Fisher Scientific) to the culture medium (concentration, 5 μM) for 2 h before fixation and immunofluorescence analysis.

Cells were treated with 10 mM HPβCD for 24 h. Positive control wells had equivalent volumes of 5 mM MβCD (Sigma-Aldrich, UK). Following treatment, cholesterol was stained with several fluorescent dyes and quantified. Namely, Filipin (Sigma-Aldrich, UK) for free cholesterol, CholEsteryl BODIPY™ FL C12 (Thermo Fisher Scientific, USA) for cholesteryl esters (CEs), and the Vybrant™ Alexa Fluor™ 594 Lipid Raft Labelling Kit (Invitrogen™, Thermo Fisher Scientific, UK) for lipid rafts. Nuclei were stained with either DAPI (Sigma-Aldrich, UK) or NucRed^® Dead 647 ReadyProbes^® Reagent (Thermo Fisher Scientific, USA). Mice tumours from three different groups were sectioned, dewaxed, and stained with Filipin as mentioned in results. Cell/tumour sections were visualised using the FLoid™ Cell Imaging Station (Thermo Fisher Scientific, Swindon, UK) and densitometry analysis was performed using the ImageJ Software (Software Version 1.41) (NIH, Bethesda, MD, USA).