This article details a method for isolating lipids from human peripheral blood-derived neutrophils using a biphasic solvent system. The process involves the use of methanol and chloroform to solubilize different lipid classes, followed by centrifugation to separate components.
Take a glass tube containing neutrophil homogenate with released intracellular components.
Add methanol, a polar solvent, that disrupts lipid-lipid and lipid-protein interactions. The inherent polarity of methanol evenly distributes polar lipids, solubilizing them. Next, add chloroform, a nonpolar organic solvent, that interacts with nonpolar lipids, causing them to disperse and solubilize.
Centrifuge to separate the protein-containing pellet from the supernatant containing lipids, polysaccharides, and cell debris. Transfer the supernatant to a fresh glass tube. Add chloroform and distilled water. Centrifuge the sample.
Water addition spontaneously forms a biphasic system, partitioning biomolecules within the lower chloroform and the upper water-methanol layers.
The chloroform layer comprises dissolved nonpolar lipids, while the upper layer contains non-lipid contaminants and more polar lipids. Remove the upper water-methanol layer without disturbing the lipid-containing layer.
Use a vacuum concentrator to remove the solvent from the lipids. Store at lower temperatures until further use.
To isolate the lipids from the human peripheral blood-derived neutrophils, take the prepared neutrophils and place them on ice. Pipette the neutrophils to a 15-milliliter screw cap glass tube with a PTFE seal, and homogenize them by shaking for one minute. Then, add 2 milliliters of methanol, followed one minute later by 1 milliliter of chloroform. After shaking for another one minute, rotate the glass tubes at room temperature and 50 RPM for 30 minutes.
Next, pellet the protein fraction by centrifuging the solution at 7 degrees Celsius and 1952 times g for 10 minutes. Carefully decant the supernatant into a new 15-milliliter glass screw cap tube leaving the protein-containing pellet behind. Store the pellet at minus 20 degrees Celsius for future quantification.
Add 1 milliliter of chloroform and wait one minute. Then, add 1 milliliter of double distilled water and invert the glass screw cap tube with the sample for 30 seconds. After centrifuging the sample as before, remove and discard the upper phase down to but not including the cloudy layer. Dry the samples in a vacuum concentrator at 60 degrees Celsius and store them at minus 20 degrees Celsius until required.
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