Milliplex panels

Multi-analyte profiling of inflammatory mediators secreted into the basolateral medium of cultures was performed using commercially available Milliplex panels (Merck Millipore) with Luminex^® xMAP^® Technology (Luminex, Austin, TX, USA)-based analysis according to the manufacturer’s instructions. Briefly, 25 µL of diluted and non-diluted sample was used for each detection and the analysis was run on the FLEXMAP 3D^® platform, equipped with xPONENT^® software version 4.2 (Luminex). Data were presented as Median Fluorescent Intensity using a five-parameter logistic or spline curve-fitting method to calculate the analyte concentrations in the basolateral medium samples. The following analytes (inflammatory mediators) were measured: chemokine [C–C motif] ligand (CCL) 20; chemokine [C–X–C motif] ligand (CXCL) 1 (also known as GRO alpha), CXCL-8 (also known as interleukin [IL]-8) and IL-6; tumor necrosis factor alpha (TNFα); soluble intercellular adhesion molecule (sICAM) 1; matrix metalloproteinase (MMP)-1 and MMP-9; tissue inhibitor of metalloproteinase (TIMP) 1, and vascular endothelial growth factor (VEGF) alpha. The data were log-transformed, and the geometric means were used to calculate the fold-changes of the mediators (exposed vs. air-exposed samples).

CCL2, CCL3 and CCL5 chemokine quantifications were conducted in mouse serum and whole skin protein extracts prepared exactly as previously described [18 (link)], using a Bio-Plex Array Reader (LUMINEX 100; Bio-Rad Laboratories, Hercules, CA, USA) and Milliplex panels (EMD Millipore, Billerica, MA, USA). Whole skin protein extracts were weighed and immediately snap-frozen using liquid nitrogen. After homogenization with a mortar and pestle, skin samples were digested for 2 hours at 4 °C in reporter lysis buffer (Promega, Madison, WI, USA) supplemented with complete mini-protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA). Supernatants were kept at −80 °C until use.