The metabolic profile was determined with commercially available colorimetric assays according to the manufacturer’s instructions. The optical density of the samples was determined with a microplate reader (Synergy 2, Biotek, Shoreline, WA, USA). The following blood serum parameters were measured by means of Pointe Scientific assays (Pointe Scientific, Canton, MI, USA): glucose (Cat. No.: G7519), triglycerides (TG; Cat. No.: T7531), total cholesterol (TCh; Cat. No.: C7510), high-density lipoprotein (HDL; Cat. No.: 7545), low-density lipoprotein (LDL; Cat No.: 7574-D) fractions, albumin (Cat. No.: A7502-500), urea nitrogen (BUN/UREA; Cat. No.: B7552-120), creatinine (Cat. No.: C7539-500), the activity of alanine aminotransferase (ALT; Cat. No.: A7526), aspartate aminotransferase (AST; Cat. No.: A7561), γ-glutamyltransferase (GGT; Cat. No.: G7571), and alkaline phosphatase (ALP; Cat. No.: A7516-120). The level of non-esterified fatty acids (NEFA) was measured with a WAKO kit (Cat No.: 434-91795 and 434-91995; Wako Chemicals, Richmond, VA, USA). The total protein concentration was measured with an Alpha Diagnostics kit (Cat. No.: A6502-100; B6528-125).
Wako kit
Manufactured by Fujifilm
Sourced in Japan, United States
Wako kits are a line of laboratory reagents and test kits produced by Fujifilm. They are designed for use in various analytical and diagnostic applications. The kits provide consistent and reliable results while maintaining high quality standards.
Automatically generated - may contain errors
Lab products found in correlation
Amplex red, by Cayman Chemical (2 mentions)
Mcp 1 ccl2, by R&D Systems (2 mentions)
Infinity kit, by Thermo Fisher Scientific (2 mentions)
Testosterone, by ALPCO (2 mentions)
Stanbio liquicolor kit, by EKF Diagnostics (2 mentions)
Il 1β, by R&D Systems (2 mentions)
Tnf α, by R&D Systems (2 mentions)
Interleukin 6 (il 6), by R&D Systems (2 mentions)
7060 automatic biochemical analyzer, by Hitachi (2 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
Enzymatic kit, by Merck Group (1 mentions)
Diagnostic kit, by Fujifilm (1 mentions)
Zorbax rx sil column, by Agilent Technologies (1 mentions)
Alliance 2695 separation module, by Waters Corporation (1 mentions)
Model 474, by Waters Corporation (1 mentions)
Beckman cx7 analyzer, by Beckman Coulter (1 mentions)
Ffa quantification kit, by Abcam (1 mentions)
Synergy neo, by Agilent Technologies (1 mentions)
21 protocols using wako kit
1
Metabolic Profiling of Blood Serum
2
Adipocyte Lipolysis Assay Protocol
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Doxycycline inducible WT PNPLA3-cherry and PNPLA3 I148M-cherry mouse brown adipocytes (BAs) were seeded on either 12 well or 24 well plates as noted. After 48 hours of induction medium, the cells were grown in differentiation media for 24 hours, followed by media containing no doxycycline or amount of doxycycline specified for a total of 4 days. After one week of differentiation, the cells were stimulated with specified treatment of either vehicle, SR-3420, or isoproterenol at the indicated concentration in H-KRBB buffer for 1 hour. Free fatty acid release was determined using WAKO kit (Wako Pure Chemicals Industries) adapted to fluorescence detection with Amplex Red (Cayman Chemical). Glycerol release was determined using glycerol reagent from triglyceride determination kit (Sigma).
3
Plasma Metabolite Levels in Cry1 Mice
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Plasma levels of glucose, triglycerides (TGs), free fatty acids (FFAs), and free cholesterol were determined in Cry1+/+ and Cry1−/− mice at the end of the 16-week HFD regimen in fed animals. Animals were sacrificed, blood was collected into EDTA-rinsed capillaries, and plasma was immediately prepared. Free cholesterol, TGs, and glucose were determined using enzymatic kits from Sigma. Plasma FFA levels were measured using the Wako kit (Wako Chemicals, Richmond, VA, USA).
4
Serum and Liver Lipid Profiling
5
Serum Biomarker Analysis in Animal Study
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Tail vein blood samples were allowed to clot on ice and then centrifuged for 10 min at 10,000g. Serum samples were stored at −80°C until analyzed. Serum and liver triglycerides (TG) and cholesterol were measured according to manufacturer's instructions using an Infinity kit (Thermo Fisher Scientific, Middletown, VA). Non‐esterified free fatty acids (NEFA) were measured using a Wako kit (Wako Chemicals, Richmond, VA). Serum β‐hydroxybutyrate (ketone) concentrations were measured with a StanBio Liquicolor kit (StanBio Laboratory, Boerne, TX). Serum insulin (Crystal Chem, Elk Grove Village, IL; cat # 90080), testosterone (ALPCO, Salem, NH; cat# 55‐TESMS‐E01), IL‐1β, IL‐6, TNF‐α, MCP‐1/CCL2 (all from R&D Systems, Minneapolis, MN; cat # DY401‐05, DY406‐05, DY410‐05, DY479‐05) levels were measured by ELISA according to manufacturer's instructions.
6
Adipocyte Lipolysis Assay Protocol
7
Intestinal Lipid Metabolism Profiling
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Small intestines were collected at 120 min after intragastric administration of triolein (10 μL/g body weight) to 12-h fasted mice, divided into three equal parts (proximal, middle, and distal) and processed as previously described (Cifarelli et al., 2017 (link)). Total triglyceride, cholesterol, and free fatty acids were determined by homogenizing 50 mg of tissue in 2 ml of chloroform:methanol (2:1 v/v). Samples were centrifuged at 12,000 rpm for 10 min at 4°C. An aliquote, 50 μl, was evaporated in a 1.5 ml microcentrifuge tube. Triglyceride and cholesterol were determined by adding 100 μL of reagent to dried lipid extracts followed by incubation for 30 min at room temperature. Level of triglyceride and cholesterol in plasma was determined using Fisher Scientific kits (Fisher Scientific, PA, United States). Non-esterified fatty acid levels in plasma were determined using Wako kit (Wako Chemicals, Richmond, VA, United States) according to the manufacturer’s protocol.
8
Fasting Blood Lipid Measurement Protocol
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Participants were required to maintain an empty stomach for 10–14 h before blood was drawn. The overnight fasting blood samples were collected by trained nurses and were stored in vacuum collection tubes. The samples were centrifuged at 3000 r/min for 10 min and the serum samples were taken, transferred to the frozen storage tube, and stored at −80 °C. The serum TC, TG, LDL-C, and HDL-C were measured by a HITACHI Automatic Analyzer (Japan, 7600-210) and Wako kit (Japan).
High TC was defined as TC ≥ 5.2 mmol/L and high TG was defined as TG ≥ 1.7 mmol/L. High LDL-C was defined as LDL-C ≥ 3.4 mmol/L and low HDL-C was defined as HDL-C ≤ 1.0 mmol/L. These were defined as borderline elevated blood lipids, according to the 2016 Chinese guideline for the management of dyslipidemia in adults [16 (link)].
High TC was defined as TC ≥ 5.2 mmol/L and high TG was defined as TG ≥ 1.7 mmol/L. High LDL-C was defined as LDL-C ≥ 3.4 mmol/L and low HDL-C was defined as HDL-C ≤ 1.0 mmol/L. These were defined as borderline elevated blood lipids, according to the 2016 Chinese guideline for the management of dyslipidemia in adults [16 (link)].
9
Triacylglycerol Measurement in Mouse Liver
10
Plasma Antioxidant Extraction and Analysis
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The plasma biochemical parameters were analyzed via colorimetric enzymatic assays with commercial kits; fasting glucose, triglycerides (TGs), and total cholesterol (TC) were assessed with Biosystems kits (Biosystems, Barcelona, Spain); and a Wako kit was used for free cholesterol (FC) (Wako Chemicals, Richmond, VA, USA). Cholesteryl esters (CEs) were calculated by subtracting the free cholesterol from the total cholesterol.
For plasma antioxidant extraction, one volume of plasma was first deproteinized with one volume of ethanol, and then, tocopherols and retinoids were extracted twice with two volumes of hexane, as previously described [15 (link)]. Tocopherols and retinoids were simultaneously separated using normal-phase HPLC with a Zorbax RX-SIL column (25 cm × 4.6 mm i.d.; 5 μm particle size; Agilent Technologies, Madrid, Spain) installed on a 2695 Alliance separation module (Waters, Barcelona, Spain) coupled with a fluorescence detector (model 474, Waters), as described previously [16 (link)].
For plasma antioxidant extraction, one volume of plasma was first deproteinized with one volume of ethanol, and then, tocopherols and retinoids were extracted twice with two volumes of hexane, as previously described [15 (link)]. Tocopherols and retinoids were simultaneously separated using normal-phase HPLC with a Zorbax RX-SIL column (25 cm × 4.6 mm i.d.; 5 μm particle size; Agilent Technologies, Madrid, Spain) installed on a 2695 Alliance separation module (Waters, Barcelona, Spain) coupled with a fluorescence detector (model 474, Waters), as described previously [16 (link)].
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