Lambda 1050 uv visible spectrophotometer

All reagents used in these experiments were purchased from Sigma-Aldrich or Tokyo Chemical Industry (TCI); their purity was 98% or higher, and they were used without further purification. Y138 was purchased from SKC Hitech and Marketing. ¹H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Advance 300 spectrometer. ¹³C NMR data were obtained from a 600 MHz AvanceIII-600 Bruker NMR equipped with a TCI Cryoprobe. Ultraviolet–visible (UV–Vis) optical absorption spectra were recorded using a Lambda 1050 UV–Visible spectrophotometer (Perkin Elmer, Waltham, MA, USA). Thermogravimetric analysis (TGA) was performed using a TA Instruments Q5000 IR/SDT Q600 with the sample under an air atmosphere. Analysis was carried out using TRIOS v 5.0 software (TA instruments, New Castle, DE, USA). Transmission electron microscopy (TEM) images were obtained using JEM-2100F (JEOL, Tokyo, Japan). The color difference, ΔE_ab, was measured using an Otsuka Electronics MCPD-3000 array spectrometer. The thickness of the color resist films was measured using a surface profiler (Dektak 150, Veeco, Plainview, NY, USA).

The amount of hydroxyproline in both treated and untreated wound tissues was determined according to the method described by Woessner (1961) (link) and modified by Gurung and Skalko-Basnet (2009) (link). Wound tissues were excised on day 13 post-injury and stored at -20°C till laboratory analysis. Samples were dried in a hot air oven at 60–70°C to constant weight and were hydrolyzed in 6 M HCl (Sigma-Aldrich, Michigan, United States) at 130°C for 3 h in sealed test tubes. The hydrolysate was neutralized to pH 7 with 2.5 M NaOH (Sigma-Aldrich, London, United Kingdom). Two milliliters of sample was transferred into a test tube and subjected to chloramine-T (Sigma-Aldrich, Michigan, United States) oxidation for 20 min at 25°C. The reaction was terminated by addition of 1 mL of 3.15 M per chloric acid (Fisher Scientific, Glasgow, United Kingdom) and allowed to stand for 5 min at 25°C. One milliliter of Ehrlich reagent (Sigma-Aldrich, St. Louis, Missouri, United States) was added, shaken, and incubated at 60°C for 20 min. The pink color developed after incubation was measured at 557 nm using a LAMBDA 1050 UV-visible spectrophotometer (PerkinElmer, 940 Winter St. Waltham, Massachusetts, United States). The amount of hydroxyproline present in the tissue sample(s) was calculated from a standard curve prepared from pure L-hydroxyproline (Sigma-Aldrich, Michigan, United States).