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