Infinity cholesterol liquid stable reagent

Liver (~50 mg) was homogenized in 500 μL of lysis buffer (250 mM of sucrose, 50 mM of Tris Cl; pH 7.4) and cOmplete Mini EDTA-free protease inhibitor cocktail (Sigma-Aldrich). Lipids were extracted using the Bligh and Danner method, resuspended in 0.1%Triton X100, and sonicated five times at 30 mA for 1 second. TG and cholesterol were measured using Infinity Triglycerides Liquid Stable Reagent and Infinity Cholesterol Liquid Stable Reagent kits (Thermo Fisher Scientific).

For cholesterol determinations, two flies were washed in Danieau’s solution pH 7.2 (17.4 mM NaCl, 0.21 mM KCl, 0.12 mM MgSO₄+H₂0, 0.18 mM Ca(NO)₃, 1.5 mM HEPES) and homogenized in 200 μl of PBST containing 0.05% Tween 20 (Apex Bioresearch Products, Genesee Scientific El Cajon, CA #18–173). Total cholesterol was measured in triplicate using the Infinity Cholesterol Liquid Stable Reagent (Thermo Fischer Scientific Waltham, MA #T1213421). Cholesterol was quantified according to manufacturer’s instructions and were read using a SpectraMax i3 plate reader (Molecular Devices, San Jose, CA). To determine triglycerides, single flies were washed in Danieau’s solution and homogenized in 200 μL PBST. Samples were diluted 1:10 in PBST and total triglyceride was measured in triplicate using the Infinity Triglyceride reagent (Thermo Fischer Scientific Waltham, MA #TR22421). Triglyceride was quantified according to manufacturer’s instructions using a SpectraMax i3 plate reader. Glucose was determined using single flies washed in Danieau’s solution and homogenized in 500 μl of 100 mM maleate buffer, pH 5.5 (Alfa Aesar Haverhill, MA #J62049). Total glucose was measure in triplicate using the Glucose Oxidase Liquid Stable Reagent (Thermo Fischer Scientific Waltham, MA TR15221). Glucose was measured according to manufacturer’s instructions and read using a SpectraMax i3 plate reader.

Mouse hippocampal slices were prepared and treated with SV or Veh for 2 and 4 h. The hippocampal area was dissected and homogenized in a tissue lysis buffer (PBS with 5 mM sodium deoxycholate, 0.1 % Triton X-100, and complete protease and phosphatase inhibitor cocktails). The cholesterol level in the tissue lysate was measured by the Infinity™ Cholesterol Liquid Stable Reagent (Thermo Scientific, Middletown, VA). The cellular cholesterol content was normalized by the cellular protein concentration measured by the BioRad Protein Assay (BioRad, Hercules, CA).