Quantification of Cellular Phospholipids

Total cellular lipids were extracted by chloroform/methanol extraction. TLC was performed as previously described [14 (link)]. Lipids were loaded onto a TLC plate based on protein quantification. Brain PS (Avanti Polar Lipids) and sphingomyelin (Matreya LLC) were run as molecular standards. Bands corresponding to PS were scraped and subjected to phosphorus extraction by acidic digestion. The liberated phosphorus was estimated by allowing a complex formation with ammonium molybdate (Sigma) and malachite green (Sigma) and by measuring the absorption at 660 nm [54 (link)]. Phosphorus was quantified using a standard curve obtained from phosphorus liberated from known concentrations of Brain PS run on TLC plate. Cellular PS was expressed as the ratio of phosphorus obtained from PS and phosphorus obtained from total phospholipids. Additionally, PS was estimated by acquiring TLC band intensities of PS and sphingomyelin, using Image Studio Lite software; variations in PS are shown as a ratio of PS to sphingomyelin bands.