Fig. 1.

Methodology-work flow for isolation of lipids and oxysterols from cellular and subcellular compartments. Three cell types - THP-1 monocytes, peripheral blood mononuclear cells (PBMC) and SH-SY5Y neuroblastoma cells-were used for this analysis. The work flow involves stage A - isolation of mitochondria from whole cells, stage B - sample lysis and lipid extraction-cholesterol and protein measurements, stage C - oxysterol isolation using solid phase extraction (SPE) followed by LC-MS/MS profiling using a multistep gradient of solvent A - 70% methanol (MeOH) + 30% H2O + 0.1% formic acid and solvent B - 90% isopropanol (IPA) + 10% MeOH and 0.1% formic acid.