Cholesterol cholesteryl ester quantitation kit

The Cholesterol/Cholesteryl Ester Quantitation Kit (Bio-Vision, Catalog: K603-100) was used to detect quantities of free cholesterol, cholesteryl esters by colorimetric methods. The Cholesterol Standard was diluted with Cholesterol Assay Buffer and added to wells generating 0, 1, 2, 3, 4, and 5 μg/well of the Cholesterol Standards, plotted the Cholesterol Standard Curve.
For the cell samples (72 hpi), 10⁶ cells could be extracted with 200 μl of chloroform: Isopropanol: NP-40 in a micro-homogenizer. Spin the extract in the centrifuge. Transfer all the liquid to a new tube, air dry to remove chloroform, and trace organic solvent. Dissolve dried lipids with 200 μl of Cholesterol Assay Buffer. The progeny virions (72 hpi) were diluted 10-fold in the Cholesterol Assay Buffer. After incubation at 37°C for 60 min in dark conditions, the absorbance of the samples was measured at 570 nm for the colorimetric assay, and the total cholesterol concentrations were calculated by comparing to those of the Cholesterol Standards.

The concentration of total cholesterol and free cholesterol released by HUVECs was determined using the commercial kit, Cholesterol/Cholesteryl Ester Quantitation Kit (Biovision, California, USA), according to the instruction described by the manufacturer [19 (link)].

Liver glycogen content of female mice was measured using the Glycogen Colorimetric Assay (BioVision, Milpitas, CA, USA). Liver homogenates (described above) were diluted 1:10 in the manufacturer’s hydrolysis buffer and 50 µL was assayed in singlet for each mouse (n = 7–9 mice/genotype). Hepatic and serum cholesterol levels were assayed in female mice using the Cholesterol/Cholesteryl Ester Quantitation Kit (BioVision, Milpitas, CA, USA). Samples were 3 µL serum and 50 µL liver homogenate per mouse (n = 7–9 mice/genotype).

Plasma insulin was determined with an Ultra-Sensitive Mouse Insulin ELISA Kit (Crystal Chem, Inc.) following the manufacturer’s protocol. Plasma FFA was measured with a NEFA C Test Kit (Wako Chemicals, Richmond, VA). Plasma triglycerides were analyzed using plasma Triglyceride Determination Kit (Sigma-Aldrich, St, Louis MO) and plasma cholesterol was determined by a Cholesterol/Cholesteryl Ester Quantitation Kit (BioVision, Milpitas, California). Plasma IL-6 and myostatin levels were measured by using ELISA Kit (EMD Millipore). Plasma irisin was determined using a mouse irisin ELISA Kit (Phoenix pharmaceutical, Burlingame, CA).

The triglyceride (TG) and cholesterol contents in the heart extracted by a chloroform/methanol (2:1) mixture were determined using the TG Assay Kit (Biovision Inc. Mountain View, CA, USA) and a Cholesterol/Cholesteryl Ester Quantitation Kit (Biovision Inc.).