No red

Owing to its instability, the quantitative analysis of NO production in/around the skin wounds was performed by simultaneously measuring the NO metabolites of nitrite (NO₂⁻) and nitrate (NO₃⁻) in the microdialysate. The method for measuring NO₂⁻ and NO₃⁻ levels was described previously47 (link). Briefly, 10 μL of the microdialysate fraction was injected into an ENO-30 NO-detector (Eicom, San Diego, CA). The NO₂⁻ and NO₃⁻ in the microdialysate were separated by a reverse-phase separation column (NO-PAK, 4.6 × 50 mm, Eicom, San Diego, CA), and NO₃⁻ was reduced to NO₂⁻ in a reduction column (NO-RED, Eicom, San Diego, CA) at 35 °C column temperature. Then, the NO₂⁻ was reacted with Griess reagent, which was delivered at a rate of 0.1 mL/min, to form a purple azo dye in a reaction coil at 35 °C. The absorbance of the product dye was detected at 540 nm by a flow-through spectrophotometer (NOD-10, Eicom, San Diego, CA). The mobile phase was purchased from Eicom and delivered at a rate of 0.33 mL/min. The concentrations of NO₂⁻ and NO₃⁻ in the microdialysate were obtained using a standard curve prepared from known concentrations of sodium nitrite and sodium nitrate. The resulting values were converted into percentages, taking the values for samples from diabetic rats without skin wounds as 100%. All experiments were performed with n = 6.

For the measurement of PGI₂, the plasma concentration of its stable metabolite 6-keto-PGF_1α was determined using a commercially available ELISA kit according to the manufacturer’s instructions.
The concentration of nitrite and nitrate were measured using ENO-20 –NOx Analyzer (Eicom Corp., Kyoto, Japan). The ENO-20 uses a liquid chromatography method with post-column derivatisation using Griess reagent. Nitrite and nitrate were separated from other substances in matrices on an NO-PAK column, 4.6x50mm (Eicom Corp.). Nitrate was reduced to nitrite using a cadmium-copper column (NO-RED, Eicom Corp.). Nitrite was mixed with Griess reagent to form a purple azo dye in a reaction coil placed in a column oven at 35°C, and the absorbance of the dye product was measured at 540 nm. The flow of the mobile phase (Carrier Solution) was 0.33 ml·min^-1. The Griess reagent (Reactor A and B Solution) was delivered by the pump at a rate of 0.11 ml·min^-1. The plasma sample was precipitated with methanol at a ratio of 1:1 (v/v), and centrifuged at 10 000 x g for 10 min, and the supernatant was used for analysis.