2.1. Cell culture and oxysterol extraction

Blood was collected from healthy individuals with approval from ethics committee. PBMC isolation and cultivation of THP-1 human monocytic cell line and SH-SY5Y cells in their respective growth medium were performed as described in the related article [1]. THP-1 monocytes were treated with 100 ng.mL⁻¹ phorbol myristate acetate for three days to differentiate them into macrophages prior to cell harvest and oxysterol analysis.

The detailed methodology for cell lysis and oxysterol extraction is described in the related article [1]. 22SOHC-D⁷ was used as the external standard in preparation of standards and samples. Briefly, whole cells and mitochondrial fractions from different cell types were sonicated in the lysis buffer (Triton, DMSO and butylated hydroxytoluene). Polar and non-polar metabolites were separated using biphasic solvent extraction with methanol, dichloromethane and water. Samples were spun and lower non-polar phase containing lipids, cholesterol and oxysterols were dried under N₂ gas, followed by solid phase extraction (SPE) to isolate oxysterols [1].