Extracting cholesterol and lipids from cells

Contributed by Martin Fitzpatrick, University of Birmingham, United Kingdom

This a general method that may not work for all cell/tissue samples - tweak the solvent volumes per quantity of cells, centrifugation time and sonication may be applied for samples that are difficult to disrupt. For chloroform - handle under a chemical safety hood.

  • Homogenize 1 x 10e6 cells or ~10 mg tissue into either 200 uL chloroform-methanol (v/v 2:1) or 200 uL hexane-isopropanol (v/v 3:2).
  • Centrifuge for 5-10 min at 14,000 rpm in a microcentrifuge.
  • Transfer the organic phase to a clean tube and vacuum dry. Store the material in the freezer (<20oC), desiccated and protected from air, i.e., under anaerobic conditions to minimize oxidation.
  • Re-dissolve the vacuum-dried lipids/cholesterol into a suitable assay buffer prior to use.