Quantifying Chemokine Levels in Mouse Skin

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.