Effect of Topical Insulin Application on Wound Neutrophil Function
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These samples were later examined by Western blotting, real-time polymerase chain reaction (PCR), myeloperoxidase (MPO), and malondialdehyde (MDA) biochemical quantification assays.
Healing times and healing rates. The area without epithelium was identified as the wounded surface, and the period of time between the injury and total epithelium recovery was identified as the wound healing time (n = 6). The wounded surfaces were drawn on transparent tracing paper and scanned for analysis. The pictures were analyzed with ImageJ software to calculate the area of the wounded surface.
Western blot assay for Gr-1 expression. Tissue samples from the wounded area were lysed with RIPA lysis buffer. Total protein was detected by bicinchoninic acid assay (BCA). A total of 50 µg of total protein from each sample was mixed with sample buffer and loaded into each well of a sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gel. Samples were then run by SDS-PAGE on 12% gradient gels, and transferred to polyvinylidene fluoride (PVDF) membrane. Transferred PVDF membranes were blocked by 5% BSA in tris-buffered saline and Tween 20 (TBST) at room temperature for 1 hour, followed by overnight incubation with a primary anti-Gr-1 antibody at 4˚C. On the second day, membranes were washed 3 times and then incubated in secondary antibody for 2 hours at room temperature. Visualization was processed by ECL reagents and compatible scanners. Blots then were re-probed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to show equal loading. Band intensities were analyzed using ImageJ software.
Biochemical analysis of MPO and MDA expression. The experimental protocol followed the manufacturer’s instructions. The unit of MPO quantification was units/g (wet sheet). The unit of MDA quantification was nmol/mg (protein amount).
Real-time quantitative PCR assay for MIP-2 mRNA expression. Total mRNA was extracted using Trizol (Life Technologies, Grand Island, NY). The ratio of OD 260 nm to OD 280 nm was detected. If the values were higher than 1.8, RNA samples were reverse transcribed. The system volume of reverse transcription was 20 µL containing 1 µg RNA. The reaction conditions of the reverse transcription were 37°C for 15 minutes and 80°C for 5 seconds. The system volume of the real-time PCR was 20 µL containing 10 µL of SYBR Premix Ex Taq™ II (2x), 8 µL of PCR forward primer (10 µm), 8 µL of PCR reverse primer (10 µm), 0.4 µL of ROX Reference Dye or Dye II (50x), 2 µL of cDNA solution, and 6.8 µl of dH2O. The primers for MIP-2 and GAPDH were designed by BioTNT (Wanchai, Hong Kong). The reaction conditions of real-time PCR were 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds, and 60°C for 30 seconds.
The ∆∆C(T) method, as described previously, was use for data analysis.18 Data were reported as mean ± standard deviation.