Overview
This video demonstrates lipid isolation from human neutrophils by a biphasic solvent system using methanol and chloroform.
Protocol
1. Lipid Isolation and Analysis of Human Blood-derived Neutrophils
- Take neutrophils and place them on ice.
- 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.
- Add 2 mL of methanol followed 1 min later by 1 mL of chloroform. Shake again for 1 min.
- Rotate the glass tubes at RT and 50 rpm for 30 min.
- Pellet the protein fraction by centrifuging the solution at 7 °C and 1,952 x g for 10 min.
- 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.
- 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.
- 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.
- If required, perform an optional further purification step by repeating step 1.1.8.
- Dry the samples in a vacuum concentrator at 60 °C and store them at -20 °C until required.
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Materials
Name | Company | Catalog Number | Comments |
10 µl syringe | Hamilton | 701 NR 10 µl | |
Cannula 26G | Braun | 4657683 | |
Chloroform | Carl Roth | 7331.1 | |
Methanol | Carl Roth | 7342.1 | |
Water | Carl Roth | 3255.1 | Endotoxin-free |