Isolation of Lipids from Human Blood-Derived Neutrophils by Biphasic Separation

1. Lipid Isolation and Analysis of Human Blood-derived Neutrophils

1. Take neutrophils and place them on ice.
2. On the ice, pipette the neutrophils (present in methanol and chloroform solution) to a 15 mL screw-cap glass tube with a polytetrafluoroethylene (PTFE) seal and homogenize them by shaking for 1 min. Use glass tubes to prevent the lipids from binding to plastic surfaces. Use PTFE caps to prevent contamination from rubber/plastic.
3. Add 2 mL of methanol followed 1 min later by 1 mL of chloroform. Shake again for 1 min.
4. Rotate the glass tubes at RT and 50 rpm for 30 min.
5. Pellet the protein fraction by centrifuging the solution at 7 °C and 1,952 x g for 10 min.
6. Carefully decant the supernatant into a new 15 mL glass screw-cap tube, leaving the protein-containing pellet behind. Store the pellet at -20 °C for future quantification.
7. Add 1 mL of chloroform, wait 1 min, add 1 mL of double-distilled water, and invert the glass screw-cap tube with the sample for 30 s.
8. Centrifuge at 7 °C and 1,952 x g for 10 min and discard the upper phase, down to, but not including the cloudy layer.
9. If required, perform an optional further purification step by repeating step 1.1.8.
10. Dry the samples in a vacuum concentrator at 60 °C and store them at -20 °C until required.