Hydroxyproline assay kit mak008

For hydroxyproline measurement, tissues were weighed and taken into eppendorf tubes and 100 µl distilled water was added. After 100 µl and 12M concentrated hydrochloric acid was added, they were kept in a laboratory oven (Thermo Scientific Heratherm oven OGS60, Langenselbold, Germany) at 120^οC for 3 hours. Cooled samples were centrifuged at 4000 rpm for 10 min. The supernatants obtained were transferred to eppendorf tubes readily prepared for activity measurement and hydroxyproline was measured spectrophotometrically at 560 nm as defined in the kit prospectus (Sigma-Aldrich® Hydroxyproline Assay Kit MAK008, St. Louis, USA). The results were determined as μg/mg ww (wet weight).

For OHP measurement, 10 mg of tissue from the snap-frozen anastomosis was shredded and hydrolyzed in 37% HCl for 3 h at 120 °C. After centrifugation, the supernatant was analyzed by chloramine T using the Hydroxyproline Assay Kit MAK008 from Sigma-Aldrich (Steinheim, Germany). Then, 20 µL lysate was added into a 96-well plate. Standards were run in parallel. All samples were run in duplicate following the manufacturers protocol. Analysis was performed using the Glomax Multi Detection System at 560 nm absorption. Results were normalized for protein content which was determined by the Bradford method.