After fasting overnight, mice were anesthetized briefly with 5% isoflurane (Abbott Animal Health) inhalation and 200 μl of blood were collected by retro-orbital plexus using a noncoated hematocrit glass capillary (Chase Scientific Glass). Blood was allowed to clot at 4°C overnight and then centrifuged at 5,000 g for 15 min (4°C). The obtained supernatant was centrifuged again at 11,000 g for 2 min (4°C). Serum total cholesterol, LDL-C, and HDL-C were measured using commercial colorimetric assays (Wako Cholesterol E, Wako L-type LDL-Cholesterol, Wako HDL-Cholesterol E; Wako Diagnostics) according to the manufacturer’s instructions.
Wako cholesterol e
Manufactured by Fujifilm
Sourced in Japan
The Wako Cholesterol E is a laboratory equipment product designed to measure the cholesterol levels in a sample. It provides a quantitative analysis of total cholesterol concentration.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Wako l type ldl cholesterol, by Fujifilm (2 mentions)
Wako hdl cholesterol e, by Fujifilm (2 mentions)
Wako nefa hr2 reagents, by Fujifilm (1 mentions)
Wako l type tg m, by Fujifilm (1 mentions)
Super glucocard 2, by Arkray (1 mentions)
Wako cholesterol e kit, by Fujifilm (1 mentions)
Nefa c test wako, by Fujifilm (1 mentions)
Tg e test wako, by Fujifilm (1 mentions)
Microplate reader, by Tecan (1 mentions)
Hdl and ldl vldl quantification kit, by Merck Group (1 mentions)
Isoflurane, by Abbott (1 mentions)
Free cholesterol e, by Fujifilm (1 mentions)
L type triglyceride m, by Fujifilm (1 mentions)
9 protocols using wako cholesterol e
1
Serum lipid profiling in mice
2
Effects of FRG on ob/ob Mice Metabolism
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A total of 18 female mice were used. The animals were divided into three groups (n = 6 mice in each group). Group I animals received drinking water (filtered tap water) and served as the untreated control group. Group II animals received drinking water containing 0.5% FRG and Group III mice received drinking water with 1% FRG for 16 wk. The effect of FRG on ob/ob mice was determined by measuring changes in body weight biweekly. Blood glucose levels were measured using a glucometer (Super Glucocard II, ARKRAY INC., Kyoto, Japan) after 3 h of fasting. Serum triglycerides were measured using the Wako L-Type TG M (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and serum total cholesterol levels were measured using the Wako Cholesterol E (Wako Pure Chemical Industries, Ltd.). Serum free fatty acids levels were measured by an enzymatic colormetric method using Wako NEFA-HR2 reagents (Wako Pure Chemical Industries, Ltd.). Finally, the animals were sacrificed, and the liver and skeletal muscle (quadriceps) were quickly removed for RNA isolation.
3
Lipoprotein Profile Analysis in Mice
4
Cellular Cholesterol Quantification
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For lipid extraction, 3 × 106 cells were plated into a 10 mm dish. Following overnight incubation for cell adherence, cells were washed twice with warm PBS and preincubated for 72 h in DMEM supplemented with 10% lipoprotein-depleted serum, containing 50 μg/ml of LDL. Cells were then washed twice with ice-cold PBS, and total lipids were extracted into hexane:isopropanol (3:2) for 30 min at room temperature. The extraction procedure was repeated three times. The combined solvents containing extracted lipids were transferred to an Eppendorf tube and dried under nitrogen. The lipid residue was resuspended in 200 μl of 5% Triton X-100. TC was measured by enzymatic colorimetric method (Wako Cholesterol E; Wako, Japan), subtracting a 5% Triton X-100 blank to eliminate nonspecific absorbance. Protein residue, precipitated after lipid extraction, was solubilized with 0.1 N NaOH, and protein concentration was determined using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). Cellular cholesterol content data were expressed as microgram cholesterol/milligram of protein.
5
Biomarker Measurements in Metabolic Study
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Blood glucose was measured using a glucometer (OneTouch Verio® IQ; LifeScan Japan Co., Ltd., Tokyo, Japan). Serum total cholesterol, non-esterified fatty acid, and triglyceride (TG) concentrations were determined with Wako Cholesterol E, NEFA C-Test Wako, and TG E-Test Wako (Wako Pure Chemical Industries, Ltd., Osaka, Japan), respectively. Insulin (Morinaga Institute of Biological Science, Inc., Kanagawa, Japan), leptin (FUJIFILM Wako Shibayagi Co., Gunma, Japan), high mobility group box 1 (HMGB1) (FUJIFILM Wako Shibayagi Co.), and fatty acid binding protein 4 (FABP4) (MyBioSource, Inc., CA, USA) concentrations were measured with an enzyme-linked immune sorbent assay kits.
6
Plasma Cholesterol Quantification in Mice
7
Serum Lipid Profiling in Mice
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After fasting overnight, mice were anesthetized briefly with 5% isoflurane (Abbott Animal Health, Abbott Park, IL) inhalation and 200 μl of blood were collected by retro-orbital plexus using a noncoated hematocrit glass capillary (Chase Scientific Glass, Rockwood, TN). Blood was allowed to clot at 4°C overnight and then centrifuged at 5,000 g for 15 min (4°C). The obtained supernatant was centrifuged again at 11,000 g for 2 min (4°C). Serum total cholesterol, LDL-cholesterol (LDL-C), and HDL-C were measured using commercial colorimetric assays (Wako Cholesterol E, Wako L-type LDL-Cholesterol, Wako HDL-Cholesterol E; Wako Diagnostics, Richmond, VA) according to the manufacturer’s instructions. HDL-C levels were measured in plasma from bone marrow (BM)-transplanted mice by using HDL and LDL/VLDL quantification kit (Sigma-Aldrich) following the manufacturer’s instructions. Four volumes of blood were collected from the inferior vena cava in a tube containing 1 vol of 3.2% sodium citrate. Blood was centrifuged at 5,000 g for 15 min at room temperature and then the supernatant was centrifuged again at 11,000 g for 3 min at room temperature. Plasma was stored at −80°C until use.
8
Comprehensive Liver Lipid Extraction
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Total lipids were extracted from a large portion of the liver comprising all 4 lobes missing a small section of the left lateral lobe that was used for other analyses. The liver was ground with a tissue grinder into a homogenous slurry and ~300 mg of slurry (weighed accurately) was homogenized in 5 mL of chloroform:methanol (2:1, v/v). The samples were incubated overnight on a shaker, centrifuged (10 min, 2000 × g) and the supernatant was collected. Sodium chloride (0.9%, w/v) was added to the supernatant and the organic solvent layer was recovered and evaporated under nitrogen to obtain the weight of total lipids. For determination of TC, free cholesterol and triglyceride concentrations, total lipids were extracted from an ~300 mg section (weighed accurately) of the left lateral lobe as described above. Total lipids were weighed and re-suspended in 1.5 mL of 10% Triton X-100 in isopropanol. TC, free cholesterol and triglycerides were measured using Wako Cholesterol E (999–02601, Wako Chemicals, Richmond, VA, USA), Free Cholesterol E (993–02501, Wako Chemicals) and L-Type Triglyceride M (Wako Chemicals) kits, respectively.
9
Cellular Cholesterol Quantification Protocol
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For lipid extraction, 3 x 10 6 cells were plated into a 10 mm dish. Following overnight incubation for cell adherence, cells were washed twice with warm PBS and preincubated for 72 h in DMEM supplemented with 10% lipoprotein depleted serum, containing 50 µg/ml of LDL. Cells were then washed twice with ice-cold PBS, and total lipids were extracted into hexane:isopropanol (3:2) for 30 minutes at RT. The extraction procedure was repeated three times. The combined solvents containing extracted lipids was transferred to an Eppendorf tube and dried under nitrogen. The lipid residue was resuspended in 200 µL of 5% Triton X-100. Total cholesterol was measured by enzymatic colorimetric method (Wako Cholesterol E; Wako, Japan), subtracting a 5% Triton X-100 blank to eliminate non-specific absorbance. Protein residue, precipitated after lipid extraction, was solubilized with 0.1 N NaOH and protein concentration was determined using Pierce™ BCA Protein Assay Kit (Thermo Fisher, Waltham, MA).
Cellular cholesterol content data was expressed as mg cholesterol/mg of protein, and data was normalized to that of the control. Each extraction was performed in triplicate and each experiment was repeated independently at least 3 times.
Cellular cholesterol content data was expressed as mg cholesterol/mg of protein, and data was normalized to that of the control. Each extraction was performed in triplicate and each experiment was repeated independently at least 3 times.
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