Lipid Extraction/Quantification

A modified Bligh-Dyer extraction method was used to extract PE from the RAW 264.7 macrophage cell pellets, the A549 cell pellets, and the in vitro mixed micelle.(6) For the RAW 264.7 macrophages, cell pellets were brought up in 200 μL of PBS followed by the addition of 10 μL of 0.5 ng/μL IS. For the mixed micelle assay, 10 μL of 0.5 ng/μL IS was added to the 100 μL of micelle mix. Following IS addition for both micelles and cell pellets, 1 mL of methanol and 0.5 mL of chloroform was added. Samples were then sonicated and transferred to the 48 °C water bath for 8 hours. Samples were then sonicated and centrifuged at 2500 G for 10 min. The supernatant was transferred to a new tube, dried down, and re-suspended in 500 μL of methanol. Samples were again centrifuged at 2500 G for 10 min, transferred to injection vials, and were submitted for PE mass spectrometry analysis on the QTRAP 5500 LC-MS/MS. Data analysis was performed using Analyst Software. Quantification of unknown peaks was done using the following formula: [(Analyte Concentration/Analyte Signal) = (F)(Internal Standard Concentration/Internal Standard Signal)] where F is signal response factor (determined using calibration curves) used to account for ionization differences between the internal standard and analytes.