Lipoprotein Separation and Cholesterol Quantification

Lipoprotein separation for total cholesterol and triglycerides estimation in plasma samples was performed using fast-protein liquid chromatography (FPLC) as described elsewhere (27 (link)). Briefly, mice were fasted 4 h, and blood was collected in anticoagulant (EDTA)-coated tubes through the submandibular plexus. Samples were centrifuged at 2500 g for 10 min at 4°C, and plasma was collected and the total cholesterol was measured (using an enzymatic kit) to ensure that pooling of samples used plasma with comparable sterols to exclude any outliers with any dramatic differences between individual mice (although no samples needed to be excluded). Column chromatography (10/300GL, Superose 6) was then used for separation of different lipoproteins by loading a minimum of 100 μl of pooled plasma mixed with 100 μl of PBS, from each group, onto the Akta pure FPLC, and a total of 52 fractions each with volume of 500 μl were collected and analyzed for cholesterol content using Infinity cholesterol (TR13421) and triglyceride (TR22421) calorimetric kits procured from ThermoFisher Scientific, Middletown, VA.