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’