Hydroxyproline proline measurement was performed according to (Stoilov et al., 2018 (link)) In brief, depilated skin tissues (~20 mg each) were hydrolyzed with 6 N hydrochloric acid (Thermo Fisher) at 105°C for 48 h, and then were dried 65°C for 90 min in SpeedVac. Samples were dissolved in 400μl H2O and vortexed vigorously. Then, an aliquot of dissolved sample was oxidized with 1.5 mg of chloramine-T (Sigma) for 20 min at room temperature followed by reaction with 20 mg of p-dimethylaminobezaldehyde (Fluka) that was dissolved in propanol containing 20% perchloric acid (Aldrich) for 10 min at 65°C. Samples were aliquot in 96 wells and read using Cytation 5 Cell Imaging Multi-Mode Reader (BioTek) at 550nm. Hydroxyproline content was determined by extrapolation from a hydroxyproline standard curve (Sigma). Hydroxyproline content per tissue weight was calculated by normalizing with tissue weight.
Hydroxyproline standard curve
Manufactured by Merck Group
The Hydroxyproline standard curve is a laboratory tool used to quantify the concentration of hydroxyproline, a amino acid commonly found in collagen. It provides a standardized reference to measure the amount of hydroxyproline present in a sample through spectrophotometric analysis. The standard curve serves as a calibration tool to determine the concentration of hydroxyproline in unknown samples.
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Lab products found in correlation
Chloramine t, by Merck Group (1 mentions)
Cytation 5 cell imaging multi mode reader, by Agilent Technologies (1 mentions)
Perchloric acid, by Merck Group (1 mentions)
Chondroitin sulfate standard, by Merck Group (1 mentions)
1 9 dimethylmethylene blue assay, by Merck Group (1 mentions)
6 n hcl, by Merck Group (1 mentions)
Papain, by Merck Group (1 mentions)
2 protocols using hydroxyproline standard curve
1
Hydroxyproline Quantification in Skin
2
Quantifying Extracellular Matrix Components
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AF and NP tissues were isolated from IVDs and immediately frozen at -80°C and lyophilized to determine dry weight. Lyophilized samples were then digested in papain solution (100mM Na2PO4, 5mM N-Acetyl-L-Cysteine, 5mM EDTA, 3.875 U/mL papain (Sigma-Aldrich)) at 65°C overnight. Digestate was then assayed for sulfated-GAG content using 1,9-Dimethyl-Methylene Blue assay with a chondroitin sulfate standard (2.5-25 µg/mL; Sigma-Aldrich). (34) To quantify collagen content, digestates were hydrolyzed for 1 hour at 121°C and 15 psi in 6N HCl (Sigma-Aldrich). Hydrolyzed digestates then underwent hydroxyproline assay as described by Cissell et al, (35) in conjunction with a hydroxyproline standard curve (5-35µg/ml; Sigma-Aldrich). Collagen content was inferred from hydroxyproline quantity using hydroxyproline : collagen percentage of 13.5%. (36) Sulfated-GAG and collagen content were respectively normalized to dry weight and were additionally expressed as the ratio of sulfated-GAG to hydroxyproline (GAG:HyPro).
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