Cholesterol cell based detection assay kit

Filipin staining was performed using the Cholesterol Cell-Based Detection Assay Kit (Cayman Chemical 10009779) and protocol. Cells were immediately imaged at a constant exposure time to avoid confounding effects from photobleaching. The experiment was repeated for each condition three independent times, yielding the same results each time as shown in Fig. 2A–D. Images selected for apoptosis was induced by exposing cells to the lysomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH), kindly provided by Hanna Appelqvist of Linkoping University. MSDH (45 µM final concentration, 42 hr treatment length) was added to confluent fibroblasts, which had previously been subject to continuous oxidizing conditions (the same conditions as used for Fig. 1A and C) to promote LF generation and/or HPβCD treatment for 7 continuous days after reaching confluence.

Cellular cholesterol was quantified by filipin III fluorescence using the Cholesterol Cell-Based Detection Assay Kit (Cayman Chemical) as per the manufacturer’s protocol. Cholesterol depletion was performed using methyl-β-cyclodextrin (MBCD), a water-soluble oligosaccharide that binds cholesterol at the plasma membrane⁹² . Treatment with 1 mM MBCD for 48 h resulted in a moderate reduction of total cellular cholesterol in HEp-2 cells, comparable to statin treatment. Addition of a 30-min pre-treatment step with 10 mM MBCD resulted in a high reduction of total cellular cholesterol without causing cell death. Fluorescent images were quantified in CellProfiler (v2.2.0).

HepG2 and BRL3A cell lines were all from American Type Culture Collection (ATCC, Manassas, VA, USA). Human hepatocellular carcinoma cell HepG2 and rat liver fibroblast BRL3A were cultured in DMEM added with 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL). All cell lines were grown in humidified tissue culture incubator (SANYO) with an atmosphere of 5% CO₂ at 37 °C. The method concerning a high-fat medium reported by Lee et al. [36 (link)] was preferred. The free fatty acid (FFA) mixture (oleic acid/palmitic acid, 1:1) was prepared with fat-free bovine serum albumin (BSA, B2064, sigma, St. Louis, MO, USA). After attaining 70% confluence, cells were cultured with normal medium or exposed to medium containing 1 mmol/L FFA, with or without 10 mmol/L betaine, for 24 h. The level of TC in cells was measured by a cholesterol cell-based detection assay kit (No. 10009779, Cayman, Ann Arbor, MI, USA). The level of cholesterol uptake was determined by a cholesterol uptake cell-based assay kit (No. 600440, Cayman, Ann Arbor, MI, USA) according to the operation manual.