Animal studies were carried out following the guidelines for animal care approved by the Institutional Animal Care and Use Committee at the Korea Research Institute of Chemical Technology (approval number: 2020-6A-10-01). Nine-week-old male C57BL/6 mice (Orient Bio, Sungnam, Korea) were randomly divided into three groups: a control group that was fed MCS diet, the NASH model group, and the treatment group that was fed an MCD diet with once-daily oral administration of vehicle (0.5% carboxymethyl cellulose) or 30 mg/kg KRX-104130. After 4 weeks of feeding, the mice were fasted overnight, and blood and tissue samples were obtained for biochemical and histological analyses. Liver TG levels and serum concentrations of liver injury markers, including ALT and AST, were determined using a colorimetric kit (MAK266, Sigma-Aldrich, St. Louis, MO, USA) or biochemical analyzer (AU480, Beckman Coulter, Atlanta, GA, USA), respectively, according to the manufacturer’s instructions. The left lobe of the liver was fixed in 10% neutralized buffered formalin and embedded in paraffin. Histological examination was performed using hematoxylin and eosin (H&E) and picrosirius red staining of 5 µm-thick sections.
Mak266
Manufactured by Merck Group
Sourced in United States
MAK266 is a laboratory instrument designed for the analysis of chemical samples. It utilizes advanced technology to perform precise measurements and data collection. The core function of MAK266 is to provide reliable and accurate results for researchers and scientists working in various fields of study.
Automatically generated - may contain errors
Lab products found in correlation
Mak043, by Merck Group (4 mentions)
Mak044, by Merck Group (4 mentions)
Mak052, by Merck Group (4 mentions)
Mak055, by Merck Group (4 mentions)
Mak045, by Merck Group (3 mentions)
Ab65359, by Abcam (3 mentions)
Mlb00c, by R&D Systems (2 mentions)
Dy479 05, by R&D Systems (2 mentions)
Mak043, by Abcam (2 mentions)
Dz042a, by Merck Group (2 mentions)
Ab277390, by Abcam (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Au480, by Beckman Coulter (1 mentions)
Concentrator plus, by Eppendorf (1 mentions)
Sta 384, by Cell Biolabs (1 mentions)
Adiponectin, by R&D Systems (1 mentions)
Infinite f200 pro, by Tecan (1 mentions)
Leptin, by BioVendor (1 mentions)
Colorimetric assay kit, by Merck Group (1 mentions)
Ke10048, by Proteintech (1 mentions)
31 protocols using mak266
1
Evaluating NASH Treatment Efficacy in Mice
2
Plasma Triglyceride Quantification
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Peripheral blood was collected as described above and plasma was used to quantify triglyceride concentrations following manufacturer’s recommendations (Sigma-Aldrich, MAK266).
3
Quantifying Circulating Triglycerides Post-Sprint
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Circulating triglycerides were assessed immediately post-sprint exercise testing. Blood was collected by cardiac puncture and left undisturbed at room temperature for 30 min. After centrifugation at 4 °C for 10 min at 2000g, the serum supernatant was collected. Triglycerides were detected from serum using a commercially available triglyceride assay kit (Sigma, MAK266). Triglyceride concentration was determined by enzymatic breakdown to glycerol. The colorimetric reaction from glycerol oxidation was detected at 570 nm in a microplate reader (VersaMax).
4
Triglyceride Quantification in Transfected Cells
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Triglycerides were extracted 48 h post-transfection using the Folch extraction method (25 (link), 26 (link)) as follows: cells were collected and homogenized in 1.5 ml chloroform:methanol (2:1) and left on ice for 30 min with occasional vortexing. This was followed by the addition of 0.38 ml water, and the mixture was left for 10 min on ice. The mixture was then centrifuged at 400xg for 5 min. The lower TG layer was collected and evaporated under vacuum using an Eppendorf Concentrator plus (Eppendorf, Germany), and then TGs were dissolved in 5% NP-40 and quantified using a triglyceride quantification colorimetric/fluorometric kit (MAK266, Sigma Aldrich, United States) following the manufacturer's instructions. The protein layer found in the interphase was collected and quantified using modified Lowry protein assay (Applied biosystem, USA) following the manufacturer's instructions. For each sample, the TG concentration was normalized to its respective protein level.
5
Liver Triglyceride Quantification
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50–100 mg of frozen liver tissue was homogenised in 1 ml of PBS and frozen in liquid nitrogen to enable further cell lysis. Samples were thawed, centrifuged briefly (15 s at 7500 rpm) and the supernatant (including the lipid layer on top) transferred to a fresh tube. Total triglycerides were measured in homogenates according to manufacturer’s instructions (Sigma, cat: MAK266).
6
Lipid Extraction and Quantification
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7
Plasma Lipid and Protein Profiling
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Concentration of plasma lipids was measured using commercially available reagents, according to manufacturer’s instructions (HDL and LDL, Sigma-Aldrich, #MAK045; Triglycerides, Sigma-Aldrich, #MAK266).
Sandwich ELISA was performed on plasma samples for the quantitative analysis of target proteins using commercially available kits, according to manufacturer’s instructions. Adiponectin: R&D Systems, Minneapolis, MN, USA, #RRP300; Leptin: BioVendor, Brno, Czech Republic, #RD291001200R; IGF-1: Medignost, Reutlingen, Germany, #E25; BDNF Merck, Darmstadt, Germany.
Sandwich ELISA was also performed on total protein extracts from the hippocampus, for the analysis of BDNF, using kit #RAB1138 (Sigma-Aldrich).
Final absorbance was read on a microplate reader (Infinite F200 pro, Tecan, Männedorf, Switzerland).
Sandwich ELISA was performed on plasma samples for the quantitative analysis of target proteins using commercially available kits, according to manufacturer’s instructions. Adiponectin: R&D Systems, Minneapolis, MN, USA, #RRP300; Leptin: BioVendor, Brno, Czech Republic, #RD291001200R; IGF-1: Medignost, Reutlingen, Germany, #E25; BDNF Merck, Darmstadt, Germany.
Sandwich ELISA was also performed on total protein extracts from the hippocampus, for the analysis of BDNF, using kit #RAB1138 (Sigma-Aldrich).
Final absorbance was read on a microplate reader (Infinite F200 pro, Tecan, Männedorf, Switzerland).
8
Serum Lipid Measurement Protocol
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Total serum cholesterol-, LDL-C/VLDL-C, HDL-C and triglyceride concentrations were measured by a commercially available assay in accordance with instructions provided by the manufacturer (MAK043, MAK045 and MAK266, Sigma Aldrich). Assay readings were measured using a FLUOstar® Omega multi-mode plate reader using spectrophotometer (BMG Labtech, Ortenberg, Germany).
9
Hepatic Biomarker Profiling in Mice
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Hepatic levels of TG (Sigma, MAK266), cholesterol (Sigma, MAK043), glycogen (Biovision, K646-100), total BA levels (Diazyme, DZ042A), serum NEFA (Sigma, MAK044), serum ALT (Sigma, MAK052), and serum AST (Sigma, MAK055) were determined according to the manufacturer’s instructions. Mouse liver IL-1β (R&D Systems, MLB00C) and CCL2 (R&D Systems, DY479-05) were detected by commercially available ELISA kit.
10
Biochemical Serum Analysis Protocol
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Blood was collected from the heart via cardiac puncture, and serum was isolated after centrifuging at 1000g for 5 min. Aspartate aminotransferase (MAK055, Sigma-Aldrich), alanine transaminase (MAK052, Sigma-Aldrich), cholesterol (AB65359, Abcam), and TGs (MAK266, Sigma-Aldrich) were estimated according to the manufacturer protocol.
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