2.11 Scratch assay.
The migratory potential of the keratinocyte cell line NCTC2544 was assessed with a wound healing scratch assay.[30] NCTC cells were grown to confluence in 60 mm culture plates at a concentration of 2 × 105 cells in complete medium. A straight scratch was made in the confluent monolayer with a p200 pipet tip. Detached cells were removed, and the edge of the scratch was flattened by washing the cells twice with PBS. Cells were replaced with complete media and treated with increasing concentration of AuNPs-SS to monitor cell motility. Scratched areas for each sample were marked and photographed immediately and after 24 and 48 hours with an EVO digital camera (Coolpix 5400, Nikon Corporation, Japan). Using the ImageJ image processing program, the size of the denuded area was determined at each time point from the digital images. The migrated area was calculated by subtracting the wound area at time point t =24 hours and t=48 hours from t =0h.
2.12 RNA extraction, semiquantitative and quantitative PCR .
Total RNA was prepared using Tri Reagent (Sigma-Aldrich, Saint Louis, Missouri, USA), agarose gel checked for integrity, and reverse transcribed with cDNA synthesis kit (BioRad, Milano, Italy) according to manufacturer’s instructions. Selected genes were evaluated by qualitative PCR using Blue Platinum PCR Super Mix (Life Technologies, Monza, Italy). Primer sequences (IDT, TemaRicerca, Bologna, Italy) were as follows:
IL1-β Forward 5’-CCTGCAGCTGGAGAGTGTGGA-3’; Reverse 5’-AGGAGGAACGGA-GACTACCC-3’
iNOS Forward 5’ -CCC TTC CGA AGT TTC TGG CAG CAG C-3’; Reverse 5’-GGC TGT CAG AGC CTC GTG GCT TTG-3’
IL6 Forward 5’- CGG AGA GGA GAC TTC ACA CAG GA-3’ Reverse 5’- GGA GAG CAT TGG AAA TTG GGG-3’
β-actin Forward 5’ GGC ACC ACA CCT TCT ACA ATG-3’ Reverse 5’- GGG GTG TTG AAG GTC TCA AAC - 3’