The hepatic iron concentrations were measured by atomic absorption spectrometry, as described previously (15 (link)). The liver tissue was homogenized and the lipids were extracted (19 (link)), and the triglyceride levels were measured using a TGE-test Wako kit (Wako Pure Chemicals, Tokyo, Japan), according to the manufacturer's instructions. The protein concentrations were determined by the Lowry method (20 (link)) using a DC protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA).
Tg e test wako kit
Manufactured by Fujifilm
Sourced in Japan
The TG E-test Wako kit is a laboratory equipment product manufactured by Fujifilm. It is designed to perform enzymatic tests for the detection and quantification of triglycerides in biological samples. The kit provides the necessary reagents and materials to conduct these tests in a laboratory setting.
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7 protocols using tg e test wako kit
1
Liver Iron, Triglyceride, and Protein Quantification
2
Effects of High-Fat Diet and Tomato Extract in Mice
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Mice were kept in individual cages in a temperature-controlled room at 23 ± 1°C and maintained under a constant 12 h light/dark cycle. Male C57BL/6 mice were purchased from CLEA Japan (Tokyo, Japan). 7-week-old mice were maintained for 7 days on a normal diet (ND) (Research Diet Inc., New Brunswick, NJ, USA) and then divided into two groups of similar average body weight. Each group was maintained on a high-fat diet (HFD) containing 60% kcal fat (Research Diet Inc.) or HFD containing 1% tomato extract (section 2.3) for 10 weeks. This animal experiment was performed under free-feeding conditions. At the end of the treatment period, anesthetized mice were sacrificed by cervical dislocation, and blood and organ samples were collected. Plasma triglyceride (TG), glucose, and non-esterified fatty acid (NEFA) levels were measured using the TG E-test Wako kit (Wako), Glucose CII-test (Wako), and NEFA C-test (Wako), respectively. All animal experiments were approved by the Kyoto University Animal Care Committee (approval code: 28–76).
3
Quantitative Lipid Extraction and Analysis
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Mouse liver and tibialis anterior muscle were isolated at the end of the experiment, immediately frozen in liquid nitrogen, and lipids extracted using 1:1 isopropyl alcohol/heptane (v/v). After evaporating the solvent, lipids were resuspended in 99.5% ethanol, and TG content was measured using the TG E-test Wako kit (Wako Pure Chemical Industries).
4
Liver Lipid Extraction and Quantification
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Liver tissues were excised, rinsed with cold PBS, and homogenized in 3 volumes of water using a BioMasher II (Nippi, Tokyo, Japan). Total lipids were extracted from 280 μL of homogenate with 500 μL of 2 : 1 (vol/vol) CHCl3/CH3OH three times. The organic phase containing lipids was washed with 0.1 M KCl and evaporated to dryness. The lipids were redissolved in a small volume of 10% Triton-X100 and quantified using the TG E-Test Wako kit (Wako, Osaka, Japan). All data collected were analyzed using an unpaired t-test after one-way ANOVA testing.
5
Plasma and Liver Lipid Analysis
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The analysis of plasma AST and ALT activities and the plasma levels of TG, TC, HDL-C, and LDL-C contents were outsourced to Oriental Yeast (Osaka, Japan). To determine the TG levels in the liver, liver samples (20–30 mg) were homogenized in 200 μL of isopropanol using a BioMasher II (Nippi, Tokyo, Japan). The homogenates were centrifuged at 1100 × g for 10 min. The supernatant was subjected to a TG assay using the TG E-test Wako kit (Wako, Osaka, Japan).
6
Triglyceride Quantification Protocol
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The TG concentrations were determined using a TG E-test Wako kit according to the manufacturer's instructions (FUJIFILM Wako Pure Chemical Corporation).
7
Quantification of Bioactive Compounds
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Gallic acid monohydrate, ellagic acid dihydrate, rubusoside, olive oil (suitable for lipase assay), and the TG E-Test Wako kit were purchased from Wako (Tokyo, Japan). Most of dried plants including dried Rubus suavissimus leaves (Chinese sweet tea or Tien-cha) were purchased from Onshindo (Saga, Japan). Other reagents were of analytical grade.
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