Total collagen content in liver tissues was determined by using a hydroxyproline assay kit (MilliporeSigma). Briefly, 100–150 mg tissue per liver sample was hydrolyzed at 110°C for 20 hours in 1 mL 6N HCl per 100 mg tissue. Hydroxyproline standards and 10 μL of hydrolyzed samples were added to 96-well flat-bottom plates and evaporated to dryness on a 60°C heat block. The sediment was dissolved in 100 μL of chloramine-T/oxidation buffer mixture for 10 minutes; then 100 μL of p-dimethylaminobenzaldehyde/perchloric acid/isopropanol mixture was added to each well and incubated at 60°C for 90 minutes. Samples were cooled to room temperature, with absorbance measured at 562 nm.
Hydroxyproline assay kit
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Sao Tome and Principe, Spain
The Hydroxyproline Assay Kit is a laboratory tool designed to quantify the amino acid hydroxyproline. Hydroxyproline is a key component of collagen, a structural protein found in various tissues. The kit provides a colorimetric method to measure hydroxyproline levels, which can be useful for research applications involving collagen analysis.
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138 protocols using hydroxyproline assay kit
1
Quantifying Total Liver Collagen
2
Hydroxyproline Content Determination
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We determined the hydroxyproline content with a Hydroxyproline Assay Kit (Millipore Sigma) according to the manufacturer’s specifications. All samples were performed in biological replicates, and standards were in technical duplicates.
3
Comprehensive Plasma Biomarker Analysis
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Male plasma alcohol concentrations were measured using an Ethanol Assay Kit (catalog# ECET100; BioAssay Systems, Hayward, CA, USA) according to manufacturer’s protocol. Animals were euthanized by cervical dislocation, blood collected postmortem and plasma insulin levels determined using the Mouse Insulin ELISA kit (catalog# EMINS; Thermo-Fisher, Waltham, MA, USA), according to the recommended protocol. Levels of IL1B, IL6, INFg and TNFa were determined using commercial ELISA assays (catalog# KMC0061 and KMC0011; Thermo-Fisher, Waltham, MA, USA and catalog# Ab100689 and Ab100747; Abcam, Cambridge, MA, USA). Total cholesterol levels were determined using the Total Cholesterol Assay Kit (catalog# STA-384; Cell Biolab, Inc, San Diego, CA USA), according to the recommended protocol. The comparative levels of low-density and high-density lipoproteins were determined using a Cholesterol Assay Kit (catalog# ab65390; Abcam, Cambridge, MA, USA), according to the recommended protocol. The levels of hydroxyproline were determined using the Hydroxyproline Assay Kit (catalog# MAK008; Millipore-Sigma, St. Louis, MO, USA), following to the recommended protocol.
4
Collagen Degradation Kinetics
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Pristine collagen scaffolds and P-CSs with the same initial weight were incubated in 0.1 mg/mL type I collagenase (MilliporeSigma) at 37 °C. After 0, 5, 10, 15, 20 or 25 min, the remnant materials were collected. A hydroxyproline assay kit (MilliporeSigma) was used to quantify the collagen content in the remnant materials (n = 6). Degradation ratio was obtained using the equation: where C0 is the collagen content of samples before enzymatic hydrolysis and Ct is the collagen content in specimens that were enzymatically hydrolyzed for a designated time-period.
5
Quantifying Tissue Hydroxyproline Content
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We determined the hydroxyproline content with a hydroxyproline assay kit (MilliporeSigma) according to the manufacturer’s specifications. First, 10 mg of tissue was minced with scissors and homogenized in 100 μL of deionized water. Second, 100 μL of 12 M HCL was added to each sample and incubated at 120°C for 3 hours. Samples were centrifuged at 10,000 g for 3 minutes, and 25 μL of the supernatant was plated into a 96-well and subsequently incubated at 60°C until all liquid was evaporated. Standards and reagents were prepared as instructed in the manufacturer’s protocol. Then, 100 μL of the chloramine T oxidation buffer mixture was added to each sample and standard and incubated for 5 minutes at room temperature; 100 μL of diluted DMAB reagent was added to each sample and standard and incubated in a 60°C water bath for 90 minutes. Absorbance was read at 560 nm. All samples and standards were performed in technical duplicates.
6
Evaluating Plasma Biomarkers in Male Subjects
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We measured male plasma alcohol concentrations using an Ethanol Assay Kit (catalog no. ECET100; BioAssay Systems, Hayward, CA, USA) according to manufacturer's protocol. We determined plasma insulin levels post-mortem using the Mouse Insulin ELISA kit (catalog no. EMINS; Thermo-Fisher, Waltham, MA, USA), according to the recommended protocol. We measured levels of IL1B, IL6, and TNFα using commercial ELISA assays (catalog no. KMC0061 and KMC0011; Thermo-Fisher, Waltham, MA, USA and catalog mo. Ab100689 and Ab100747; Abcam, Cambridge, MA, USA). To determine total cholesterol levels, we used the Total Cholesterol Assay Kit (catalog no. STA- 384; Cell Biolab, Inc, San Diego, CA USA), according to the recommended protocol. To compare the levels of low-density and high-density lipoproteins, we used a colorimetric Cholesterol Assay Kit (catalog no. ab65390; Abcam, Cambridge, MA, USA), according to the recommended protocol. We used the Triglyceride Assay Kit (catalog no. ab65336; Abcam, Cambridge, MA, USA) to contrast the abundance of hepatic triglycerides, according to the suggested protocol. To compare the levels of hydroxyproline, we used the Hydroxyproline Assay Kit (catalog no. MAK008; Millipore-Sigma, St. Louis, MO, USA), and followed the recommended protocol.
7
Quantifying Muscle Collagen Content
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To assess total muscle collagen content, hydroxyproline was assayed from approximately 10 mg of muscle tissue similar to our prior methods (128 (link)) using a modified protocol with a commercially available Hydroxyproline Assay Kit (MAK008, MilliporeSigma). Details are available in the Supplemental Methods.
8
Quantifying Collagen and GAG in Decellularized Heart Valve ECM
9
Quantification of Collagen Content in Tissue Samples
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Hydroxyproline quantification was performed with a Hydroxyproline Assay Kit (MAK008, Sigma-Aldrich, Saint Louis, MO, USA), following the manufacturer’s instructions. Briefly, lyophilized native and decellularized SIS samples (≈5 mg lyophilized dry tissue) were homogenized in equal volumes of water and hydrochloric acid (12 M) and hydrolysed at 120 °C for 3 h. The supernatant of each sample was transferred to a 96-well plate and evaporated in an oven at 60 °C. Chloramine T/oxidation buffer mixture was added to each sample and incubated for 5 min at RT. Then, diluted DMAB reagent was added and incubated at 60 °C for 90 min. Absorbance was measured at 560 nm with a Spark 10M microplate reader (Tecan, Mannedorf, Switzerland). Standard curves were generated using a hydroxyproline standard solution provided by the manufacturer. Hydroxyproline content was calculated as μg hydroxyproline/mg dry tissue.
Based on the hydroxyproline quantity, the collagen content was estimated according to two methods: (i) , where the value 7.46 is the average value of the range 7.14 – 7.69 in which the hydroxyproline/collagen ratio varies; and (ii) [109 (link),110 (link)].
Based on the hydroxyproline quantity, the collagen content was estimated according to two methods: (i) , where the value 7.46 is the average value of the range 7.14 – 7.69 in which the hydroxyproline/collagen ratio varies; and (ii) [109 (link),110 (link)].
10
Quantifying Collagen and Elastin in Tissue
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Hydroxyproline (Hyp) and elastin were quantified in the NBPs and NPPs (control) and DBPs and DPPs samples. Proteins were extracted from lyophilized tissue samples (4–5 mg). Elastin was extracted using the Fastin™ elastin assay kit (Biocolor, Belfast, Northern Ireland), according to the manufacturer’s instructions. Collagen content was determined from the amount of Hyp that was extracted by the Hydroxyproline Assay Kit (Sigma-Aldrich, St. Louis, MO, USA), following the kit’s protocol. The absorbance of the final solution containing elastin and Hyp was measured at 513 nm and 560 nm with a Spark 10M microplate reader (Tecan, Männedorf, Switzerland). All samples were run in duplicate.
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