1) Hepatic enzymatic changes: Serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) were measured using ALT and AST assay kits (Lot number 20180628, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). 2) Glucose metabolism: Following 12 h of fasting, approximately 3 μL of blood was collected from the tail vein, and fasting blood glucose (FBG) was measured using a Roche blood glucose meter (Roche diagnostic GmbH, Germany). Fasting insulin (FINS) levels in the serum of mice from each group were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Ultra Sensitive Mouse Insulin ELISA Kit, lot number: 90080, Crystal Chem, USA). The homeostatic model assessment-insulin resistance (HOMA-IR) index was calculated with the following formula: FBG (mM) x FIN (IU/L)/22.5. 4) Liver triglyceride (TG) content was measured using a kit (Lot number: 2018080029, Dong’ou Diagnostic Products Co. Ltd., Zhejiang, China). 5) Liver hydroxyproline (HYP) content was measured using an HYP assay kit (lot number: 20180630, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Hyp assay kit
Manufactured by Nanjing Jiancheng
Sourced in China
The HYP assay kit is a laboratory instrument designed for the detection and quantification of hydroxyproline (HYP) levels. HYP is an amino acid that is a key component of collagen, a structural protein found in various tissues. The kit provides the necessary reagents and protocols to measure HYP concentration in biological samples, such as tissue homogenates or cell culture supernatants.
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Lab products found in correlation
Blood glucose meter, by Roche (1 mentions)
Alt and ast assay kit, by Nanjing Jiancheng (1 mentions)
Ultra sensitive mouse insulin elisa kit, by Crystal Chem (1 mentions)
Ast assay kit, by Nanjing Jiancheng (1 mentions)
Tg assay kit, by Nanjing Jiancheng (1 mentions)
Alt assay kit, by Nanjing Jiancheng (1 mentions)
Elx800, by Agilent Technologies (1 mentions)
Mda assay kit, by Nanjing Jiancheng (1 mentions)
Gsh px assay kit, by Nanjing Jiancheng (1 mentions)
Gsh assay kit, by Nanjing Jiancheng (1 mentions)
Spectramax m2e, by Molecular Devices (1 mentions)
X71 f22ph, by Olympus (1 mentions)
Microplate reader, by Thermo Fisher Scientific (1 mentions)
21 protocols using hyp assay kit
1
Hepatic Enzymatic and Metabolic Assessment
2
Quantifying Collagen Content in Bone
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The content of Hyp, the specific amino acid in collagen, was determined as an indicator of collagen content. Approximately 50 mg of bone tissues were powdered in a liquid nitrogen bath, hydrolyzed by sodium hydroxide and oxidized by chloramine-T. Then, the Hyp content was determined according to the introduction of the Hyp assay kit (Nanjing Jiancheng Bioengineering Inst., Nanjing, China).
3
Hydroxyproline and Cytokine Analysis in Pulmonary Fibrosis
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Hydroxyproline (HYP), a product of collagen hydrolysis, is one of the markers of pulmonary fibrosis. In our present study, HYP was determined by the alkaline hydrolysis method. HYP level in lung tissue was determined by a commercial HYP assay kit (Nanjing Jiancheng Bioengineering, Nanjing, China) using 100 mg of lung tissue according to the manufacturer’s instructions.
The cytokines in lung tissues were determined by ELISA assays using commercial ELISA kits (Lianke Biotech Co., Ltd., Hangzhou, China) according to the manufacturer’s instructions. Briefly, lung tissue was homogenized with PBS and centrifuged (12,000 g/min) for 10 min to obtain the supernatant and then incubated with antibodies in a microtiter plate. After incubation with HRP-conjugated streptavidin, the samples developed color in the presence of TMB and were terminated by stop solution. The OD values were detected at a wavelength of 450 nm.
The cytokines in lung tissues were determined by ELISA assays using commercial ELISA kits (Lianke Biotech Co., Ltd., Hangzhou, China) according to the manufacturer’s instructions. Briefly, lung tissue was homogenized with PBS and centrifuged (12,000 g/min) for 10 min to obtain the supernatant and then incubated with antibodies in a microtiter plate. After incubation with HRP-conjugated streptavidin, the samples developed color in the presence of TMB and were terminated by stop solution. The OD values were detected at a wavelength of 450 nm.
4
Hepatic Lipid and Collagen Assessment
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Hepatic TG content was measured using a kit (Lot number: 2018080029, Dong'ou Diagnostic Products Co. Ltd., Zhejiang, China), and hepatic hydroxyproline (HYP) content was measured using an HYP assay kit (lot number: 20180630, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), as previously described (37 (link)).
5
Hypocrellin Levels in Photoaged HaCaT Cells
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The effects of SFP and DSFPs (DSFP-15, DSFP-30, DSFP-45, DSFP-60, DSFP-75, DSFP-90, DSFP-105, and DSFP-120) on the HYP level of photoaged HaCaT cells were measured using HYP assay. HaCaT cells were seeded in 12-well plates at a concentration of 2.5 × 105 cells per well and treated according to Section 3.8.2 . The cell-free culture supernatant was collected to measure the level of HYP using the HYP Assay Kit (A030-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) in accordance with the manufacturer’s protocol.
6
Quantifying Lung Hydroxyproline Content
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Hyp content in lung tissues from the mice was determined by colorimetric assay. At 3, 6, 10, and 20 weeks, the lung tissues (n=6 mice per group) were weighed, mechanically homogenized, and snap-frozen. Hydrolysate was analyzed with a Hyp assay kit (Jiancheng, China) in accordance with the manufacturer’s instructions. The Hyp content was calculated according to the total lung weight and was expressed as micrograms.
7
Serum and Tissue Biomarker Assays
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The obtained mice blood was centrifuged at 3000 × g for 10 min, and the supernatant was collected. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), and the tissues levels of TG and hydroxyproline (HYP) were measured by using ALT assay kit (C009-2), AST assay kit (C010-2), TG assay kit (A110-1), and HYP assay kit (A030-2) following Nanjing Jiancheng Institute of Biotechnology protocols (Jiangsu, China).
8
Liver Function and Collagen Assessment
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The activities of serum alanine transaminase (ALT) and aspartate transaminase (AST), important indicators of liver function, were measured using a commercial kit (Nanjing Jiancheng Bioengineering Institute, China). The activity of hydroxyproline (HYP), reflecting the collagen concentration, were determined with an HYP assay kit (Nanjing Jiancheng Bioengineering Institute, China).
9
Hydroxyproline Quantification in Lung
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A HYP assay kit (Nanjing Jiancheng Bioengineering Institute) was used to measure the concentration of HYP in lung tissue samples. The experimental procedure was performed according the instruction. Briefly, the lung tissue (30 mg) was homogenized in 1 ml hydrolysate (95°C water bath for 20 min). After neutralizing the hydrolysates with NaOH, the mixture was diluted to 10 ml with water. Subsequently, the absorbance values of the prepared samples were detected at 550 nm. The results were expressed as μg hydroxyproline/mg wet lung.
10
Determination of Liver Injury Markers
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Kits for determining serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Total collagen content was tested by measuring the amount of hydroxyproline (Hyp) in liver tissue using Hyp assay kit obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The liver sections imbedded in paraffin were cut (5 μm) and stained with hematoxylin–eosin (H&E), Sirius Red, and Masson’s trichrome. Image J was used to determine the Sirius Red-positive area. The macroscopic examination was blindly carried out by two independent observers. AAV (type9)-control and AAV-promoter of glial fibrillary acidic protein (pGFAP)-Cyp4a14 (type9, contract number HH20181228LQJ-AAV01) were purchased from Hanbio, Shanghai, China.
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