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Bligh-Dyer lipid extraction

LI Lishann M. Ingram
MF Morgan C. Finnerty
MM Maryam Mansoura
CC Chau-Wen Chou
BC Brian S. Cummings
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Cells were washed twice, harvested in 1x phosphate buffered saline (PBS), and subsequently centrifuged. Phospholipids from cells were then immediately extracted using both chloroform and methanol/water according to Bligh and Dyer method [15]. Cell lines were suspended in 3 mL of each methanol/water and chloroform. Tubes were vortexed for 30 s and sat under the hood on ice for 10 min, followed by centrifugation (300 x g; 5 min). The bottom-most layer of chloroform was then transferred to a new test tube and spiked with a mix of commercialized SPLASH Lipidomix internal standards (Avanti Polar Lipids, Inc., Alabaster, Alabama, USA). SPLASH Lipidomix Mass Spec standards include all major lipid classes at ratios similar to those of human plasma. This extraction procedure was repeated three times and the chloroform layers from each extraction were combined. Collected chloroform layers were dried under nitrogen, reconstituted with 50 μL of methanol: chloroform (3:1 v/v), and stored at 80 °C until analysis.

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