In tissue lysates (whole brain and midbrain), we performed lipid extraction using the method of Folch. For cells, we used the Bligh and Dyer method after scraping cells with PBS [38 (link)]. To separate neutral lipids via thin layer chromatography (TLC), we employed silica gel G plates with a mobile phase consisting of hexane:ethyl ether:acetic acid (50:50:2.6, v:v). After separation, we scraped the spots corresponding to cholesterol, cholesteryl esters, diacylglycerol (DAG), and TAG from the silica and eluted them. The quantification of these lipids was carried out using the commercial kits TG color GPO/PAP AA and enzymatic Colestat (1780107 and 1009802, respectively, Wiener lab Group) following the manufacturer's instructions [10 (link)].
Colestat
Manufactured by Wiener Lab
Sourced in Argentina
About the product
Colestat is a laboratory instrument designed for the quantitative determination of cholesterol levels in biological samples. It provides accurate and precise measurements of cholesterol concentrations using a spectrophotometric method.
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7 protocols using «colestat»
1
Lipid Extraction and Quantification Protocol
2
Diurnal Glycemia and Lipidomics
Mice were restrained for taking blood samples from the caudal venous sinus, in order to measure ad libitum glycemia rhythm (at ZT3, ZT6, ZT9, ZT12, and ZT24), and delivering an IPGTT at both ZT6 and ZT18 to measure glycemia (digital glucometer Contour TS, Bayer) and plasma insulinemia responses with an ELISA kit (EMINS, Thermofisher Scientific, United States) according to manufacturer’s instructions, followed by absorbance measurement at 450 nm in Cytation 5 imaging reader (Biotek Instruments, United States).
Samples at the endpoint were taken at ZT6 and ZT18 under isoflurane (5% in oxygen, 500 ml/min) anesthesia, first by bleeding out euthanized mice by cardiac puncture, for measuring total cholesterolemia (Colestat, Wiener Lab, Argentina), HDL (HDL cholesterol, Wiener Lab, Argentina) and LDL cholesterolemia (LDL cholesterol, Wiener lab, Argentina). All measurements were performed following the manufacturer’s instructions, followed by absorbance measurements (Smartspec 3,000 UV/Vis, Biorad, United States) at 505 nm for total cholesterolemia, 600 nm for HDL, and 660 nm for LDL. A subset of mice was destined for microbiota analysis, collecting a sample of cecum content according to Tong et al. (31 (link)) at ZT0 and ZT12, in order to have one time-point at the end of the feeding phase, and the other one at the end of the fasting phase.
Samples at the endpoint were taken at ZT6 and ZT18 under isoflurane (5% in oxygen, 500 ml/min) anesthesia, first by bleeding out euthanized mice by cardiac puncture, for measuring total cholesterolemia (Colestat, Wiener Lab, Argentina), HDL (HDL cholesterol, Wiener Lab, Argentina) and LDL cholesterolemia (LDL cholesterol, Wiener lab, Argentina). All measurements were performed following the manufacturer’s instructions, followed by absorbance measurements (Smartspec 3,000 UV/Vis, Biorad, United States) at 505 nm for total cholesterolemia, 600 nm for HDL, and 660 nm for LDL. A subset of mice was destined for microbiota analysis, collecting a sample of cecum content according to Tong et al. (31 (link)) at ZT0 and ZT12, in order to have one time-point at the end of the feeding phase, and the other one at the end of the fasting phase.
3
Cholesterol and Lipid Profile Analysis
Cholesterol was determined using a colorimetric enzymatic kit (Colestat, Wiener Lab, Argentina). Further, lipid profile (LDL, VLDL, and HDL) was performed by Laboratorio Clinico de Medicina Nuclear (Santiago, Chile).
4
Serum Cholesterol Measurement Protocol
Blood drawn from a vein upon fasting for at least 10 hours was used. Total cholesterol (TC) level was measured using the enzymatic AA liquid Colestat method (Wiener Lab). Hypercholesterolemia was defined as equal to or higher than 200 mg/dL. Serum was separated within the first hour following the blood draw, and tests were done in the same day using a Metrolab 2100 autoanalyzer. The technique used to measure cholesterol was subjected to a traceability control through the Reference and Standardization Lab for Clinical Biochemistry (Laboratorio de Referencia y Estandarización en Bioquímica Clínica, LARESBIC) of the Argentine Biochemistry Foundation (Fundación Bioquímica Argentina), and the corresponding certificate was obtained from the Cholesterol Reference Method Laboratory Network (CDC, Atlanta, USA).
Cholesterol levels were defined as high (≥ 200 mg/dL), desirable (< 170 mg/dL), and moderately high (170-199 mg/dL). 14
Cholesterol levels were defined as high (≥ 200 mg/dL), desirable (< 170 mg/dL), and moderately high (170-199 mg/dL). 14
5
Serum Cholesterol Measurement Protocol
Blood drawn from a vein upon fasting for at least 10 hours was used. Total cholesterol (TC) level was measured using the enzymatic AA liquid Colestat method (Wiener Lab). Hypercholesterolemia was defined as equal to or higher than 200 mg/dL. Serum was separated within the first hour following the blood draw, and tests were done in the same day using a Metrolab 2100 autoanalyzer. The technique used to measure cholesterol was subjected to a traceability control through the Reference and Standardization Lab for Clinical Biochemistry (Laboratorio de Referencia y Estandarización en Bioquímica Clínica, LARESBIC) of the Argentine Biochemistry Foundation (Fundación Bioquímica Argentina), and the corresponding certificate was obtained from the Cholesterol Reference Method Laboratory Network (CDC, Atlanta, USA).
Cholesterol levels were defined as high (≥ 200 mg/dL), desirable (< 170 mg/dL), and moderately high (170-199 mg/dL). 14
Cholesterol levels were defined as high (≥ 200 mg/dL), desirable (< 170 mg/dL), and moderately high (170-199 mg/dL). 14
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