4β hydroxycholesterol

Methyl-β-cyclodextrin (MβCD)-cholesterol complex commercially available (Sigma, C4951). For MβCD-sterol complexation, 50 mg/mL sterol stocks in 1:1 chloroform:methanol were prepared for desmosterol, lathosterol, cholestenone, cholesterol sulfate, 4β-hydroxycholesterol (Avanti Polar Lipids), 7DHC and cholesterol (Sigma or Avanti Polar Lipids). Stoichiometry of MβCD-cholesterol complexes typically consists of one cholesterol molecule entrapped within two CDs (1:2 molar ratio). We found optimal loading conditions using a 1:7 molar ratio, where desired sterol concentration was dried under nitrogen flow, followed by addition of 5 mM MβCD (Alfa Aesar, 1303.31 g/mol) prepared in serum-free DMEM. Sterol suspensions were sonicated for 5 min and incubated at 37°C with agitation overnight. Sterol crystals were subsequently removed via 0.45 μm filtration. Sterol loading rescue experiments were performed by incubation for 1 h at 37°C supplemented with 0.5% BSA, followed by removal and addition of 7.5% LPDS for imaging or subsequent analysis to limit prolonged exposure to MβCD. Comparison of the physical properties and phase separation behavior of sterols was previously described (Bacia et al., 2005 (link); Keller et al., 2004 (link); Megha et al., 2006 (link); Wenz and Barrantes, 2003 (link)).