Fasting, plasma total cholesterol, HDL-C, triglycerides, and glucose levels were measured on a Roche Cobas 8000 modular analyzer. LDL-C was estimated by the Friedewald equation. In addition, colorimetric assays (Cholesterol Quantification Kit, MAK043, Sigma-Aldrich; Triglyceride Assay Kit, ab65336, Abcam) were used.
Cholesterol quantification kit
Manufactured by Merck Group
Sourced in United States, Macao
The Cholesterol Quantification Kit is a laboratory equipment product that enables the quantitative analysis of cholesterol levels in various samples. It provides a reliable and accurate method for measuring total cholesterol concentrations.
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Lab products found in correlation
Triglyceride quantification assay kit, by Abcam (3 mentions)
Triglyceride quantification kit mak266, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Triglyceride assay kit, by Abcam (1 mentions)
Cobas 8000, by Roche (1 mentions)
Biotek synergy neo, by Agilent Technologies (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Triglyceride colorimetric assay kit, by Merck Group (1 mentions)
Synergy 2 elisa plate reader, by Agilent Technologies (1 mentions)
Z gel microtube, by Sarstedt (1 mentions)
Mouse serum adipokine immunoassay elisa kit, by Merck Group (1 mentions)
Bio plex 200 system, by Bio-Rad (1 mentions)
Free fatty acid quantification kit, by Merck Group (1 mentions)
Triglyceride colorimetric assay kit, by Cayman Chemical (1 mentions)
Accu chek aviva, by Roche (1 mentions)
Labassay nefa kit, by Fujifilm (1 mentions)
Serum triglyceride determination kit, by Merck Group (1 mentions)
Ultra sensitive mouse insulin elisa kit, by Crystal Chem (1 mentions)
Lysosome enrichment kit, by Thermo Fisher Scientific (1 mentions)
Simvastatin, by Merck Group (1 mentions)
29 protocols using cholesterol quantification kit
1
Lipid and Glucose Biomarker Quantification
2
Cholesterol Quantification in Cells
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The extraction process was based on the method described by Bligh and Dyer [14 (link)]. Briefly, after extensive wash, the cells were pelleted and resuspended in a lysis buffer (Beyotime, China) containing 1 mM phenylmethanesulfonylfluoride (PMSF). The supernatant was collected after centrifugation at 12000 rpm for 15 min at 4 °C. Protein concentrations were measured using the Enchanced BCA Protein Assay Kit (Beyotime, China). 100 μl of lysate was placed in a microtube, and then 125 μl of chloroform and 250 μl of methanol were added. The mixture was vortexed for 1 min. The apolar and polar phases were divided by the addition of 125 μl of chloroform and 125 μl of distilled water. The mixture was centrifuged at 1000 rpm at room temperature for 5 min. The lower lipid containing phase was recovered and transferred to a clean microtube. Total cholesterol (TC) and free cholesterol (FC) contents were determined using the Cholesterol Quantification Kit (Sigma Aldrich, USA) and measured using a BioTek Synergy NEO (BioTek Instrument, USA) at the absorbance of 570 nm. CE was determined as the difference between TC and FC (TC minus FC) and was expressed in units of mg/g cellular protein.
3
Quantifying Total Cellular Cholesterol
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Total cholesterol levels were quantified using the “Cholesterol Quantification Kit” (Sigma MAK043) per manufacturer’s protocol. Briefly, equal number of cells were seeded for the experiment. Following infection, cells were harvested in 200 microlitre of solution containing chloroform-isopropanol-IGEPAL (7:11:0.1). Organic phase was separated by centrifugation at 13000rpm, followed by air drying at 50°C to remove residual chloroform. Samples were then subjected to Vacuum drying to remove any residual organic solvent. Dried lipids were dissolved in 200 microlitre of cholesterol assay buffer, vortexed and mixed. Appropriate dilutions were made in cholesterol assay buffer for the reaction in 96 well plates. Absorbance was measured at 570nm. Cholesterol standard was prepared, and results were plotted on a standard curve.
4
Plasma Cholesterol and Triglyceride Levels in Mice
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Blood was obtained by puncture of the retro-orbital plexus. Anticoagulation was performed with 0.1 volume of 136 mmol/L trisodium citrate. Subsequently, plasma was immediately isolated by centrifugation at 1100× g for 10 min and stored at –20 °C [54 (link),55 (link),56 (link)].
Plasma cholesterol and lipoprotein cholesterol levels in C57BL/6J LDLr−/− mice were determined using the Cholesterol Quantification kit from Sigma-Aldrich (Sigma, St. Louis, MO, USA). HDL and non-HDL lipoproteins were separated by ultracentrifugation as described [12 (link)]. Plasma triglyceride concentration was quantified using the Triglyceride Quantification Kit MAK266 (Sigma-Aldrich) according to the instructions of the manufacturer.
Plasma cholesterol and lipoprotein cholesterol levels in C57BL/6J LDLr−/− mice were determined using the Cholesterol Quantification kit from Sigma-Aldrich (Sigma, St. Louis, MO, USA). HDL and non-HDL lipoproteins were separated by ultracentrifugation as described [12 (link)]. Plasma triglyceride concentration was quantified using the Triglyceride Quantification Kit MAK266 (Sigma-Aldrich) according to the instructions of the manufacturer.
5
Liver Lipid Extraction and Quantification
6
Quantifying Cellular Cholesterol Levels
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Total cholesterol content of lipoprotein receptor-deficient cell lines was quantified using the Cholesterol Quantification Kit (MAK043) by Sigma Aldrich (St. Louis, Missouri, USA) according to the manufacturer’s instructions. Briefly, 1 × 106 cells were collected and frozen at −20 °C until cholesterol quantification. Lipids were extracted using chloroform:isopropanol:IGEPAL CA-630 (7:11:0.1) and the organic phase was dried under vacuum using a Savant DNA-SpeedVac concentrator (Thermo Fischer Scientifc, Waltham, MA, USA). Extracted lipids were dissolved in Cholesterol Assay buffer and stored at −20 °C. Reaction mix was set up according to the manufacturer’s instructions and absorbance was measured at 570 nm in a Biotek Synergy 2 ELISA plate reader (Winooski, VT, USA). Cholesterol amount was extrapolated from an in-run cholesterol standard curve. Blank was subtracted from all values for background correction. The final cholesterol concentration was calculated by equating the cholesterol amount to the sample volume.
7
Isolation and Characterization of HDL
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On Day 10, animals were tranquilized by intramuscular injection of Azoperone (40 mg/mL; 7 mg/kg), anaesthesia was induced by propofol (2%; 3 mg/kg), and maintained by isofluorane inhalation (3%). Animals were then sacrificed by propofol overdose and potassium chloride (2 M). HDLs were isolated by standard sequential preparative ultracentrifugation techniques from plasma-EDTA (ethylenediaminetetra-acetic acid) before sacrifice as previously reported.19 (link) Protein levels in HDL were quantified by standard Bradford protein assay (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, Waltham, Massachusetts, USA). Lipoprotein cholesterol content was assessed by the Cholesterol Quantification Kit (MAK043-Sigma-Aldrich; MO, USA). The purity of the HDL fraction was confirmed by excluding the presence of ApoB (by-agarose gel electrophoresis), microvesicles (FACS for Anexin V+ microvesicles), and exosomes (western blots for transpanins CD63 and CD81; exosomes isolated by ExoMir™ Kit).23 (link)
8
Metabolic Biomarkers in Diabetic Mice
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Fasted insulin was measured following a 6-hr fast at 18 weeks in both cohorts. Fasted blood samples were not collected from NOD mice in cohort 2, as they showed distress during fasting. Blood was collected via submandibular bleed, placed on ice for at least 30 min to allow clotting, then centrifuged at 10 × g for 5 min in 1.1 ml Z-Gel microtubes (Sarstedt). Insulin concentrations were quantified using a Mouse Serum Adipokine Immunoassay ELISA kit (Millipore, Bedford, MA) on a Bio-Plex 200 System (Bio-Rad, Hercules, CA).
Alanine aminotransferase (ALT) and cholesterol analysis was performed on serum samples taken at necropsy in both cohorts. Blood was collected via cardiac puncture, chilled for at least 30 min, then centrifuged at 10 × g for 5 min in 1.1 ml Z-Gel microtubes (Sarstedt). ALT activity was quantified in duplicate using a fluorometric ALT Activity Assay Kit per the manufacturer’s instructions (Sigma [Sigma Chemical], St. Louis, MO). Total, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) cholesterol concentrations were measured in duplicate using a colorimetric Cholesterol Quantification Kit per the manufacturer’s instructions (Sigma).
Alanine aminotransferase (ALT) and cholesterol analysis was performed on serum samples taken at necropsy in both cohorts. Blood was collected via cardiac puncture, chilled for at least 30 min, then centrifuged at 10 × g for 5 min in 1.1 ml Z-Gel microtubes (Sarstedt). ALT activity was quantified in duplicate using a fluorometric ALT Activity Assay Kit per the manufacturer’s instructions (Sigma [Sigma Chemical], St. Louis, MO). Total, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) cholesterol concentrations were measured in duplicate using a colorimetric Cholesterol Quantification Kit per the manufacturer’s instructions (Sigma).
9
Cholesterol Quantification in Tat/Cocaine-Treated Rat Neurons
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E18 rat primary neurons were cultured in 12-well plates (106/well) and were maintained 12 DIV (days in vitro) prior to the treatments. Recombinant Tat protein and cocaine (dissolved in sodium citrate buffer, pH = 5.5) were added at final concentrations of 50 ng/ml and 4 μM, respectively. This treatment was repeated every 24 hours for a total of 72 hours (15 DIV) following which cells were harvested using chloroform/isopropanol/NP-40 buffer. After solvent removal, the extracted lipid droplets under the control, cocaine, Tat and Tat\cocaine were used to determine the free and total cholesterol and CE according to manufacturer’s instructions using the cholesterol quantification kit (Sigma-Aldrich, St. Louis, MO).
10
Quantifying Lipid Metabolism in Colon Cancer
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Equal numbers of colon cancer cells were seeded in 6-well plate in regular growth medium. After 16 h incubation, cells were lysed and extracted according to procedures specified by individual quantification kits. The Free Fatty Acid Quantification Kit, Cholesterol Quantification Kit (Sigma-Aldrich), and Triglyceride Colorimetric Assay Kit (Cayman, MI, USA) were used to quantify levels of cellular fatty acids, cholesterol, and triglyceride, respectively.
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