Human il 6 and il 8 cytoset

Human IL-6 and IL-8 were investigated using ELISA kits (Human IL-6 and IL-8 CytoSetTM (CHC1263, CHC1303) Invitrogen Corporation, Carlsbad, CA, USA) following the manufacturer instructions. The amounts of cytokines were normalized to total protein content measured with Pierce 660 nm Protein Assay (Thermo Fisher Scientific Inc., Rockford, IL, USA). Absorbance was measured with EnSpire^® Multimode plate reader (Perkin Elmer, Akron, OH, USA).

IL-6 and IL-8 concentrations in skin extracts and culture media were measured using ELISA kits (Human IL-6 and IL-8 CytoSetTM (CHC1263, CHC1303) Invitrogen Corporation, Carlsbad, CA, USA) following the manufacturer instructions. The amounts of the cytokines in the skin extracts were normalized to the total protein content measured with a Pierce 660 nm Protein Assay (Thermo Fisher Scientific Inc., Rockford, IL, USA). Absorbance was measured with an EnSpire^® Multimode plate reader (Perkin Elmer, Akron, OH, USA).
The JAK inhibitory activity was measured by determining the levels of phosphorylated STAT3 and STAT5 proteins. The Human/Mouse Phospho-STAT3 (Y705) DuoSet IC ELISA was purchased from R&D System (Minneapolis, MN, USA) and the STAT5 alpha/beta (Phospho) [pY694/pY699] Human InstantOne™ ELISA Kit was purchased from Invitrogen Corporation, Carlsbad, CA, USA. For pSTAT5, the skin samples were extracted using the provided lysis buffer, whereas for pSTAT3 lysis buffer was prepared as follows: 1 mM EDTA, 0.5% Triton X-100, 5 mM NaF, 6 M Urea, 25 μg/mL Leupeptin, 25 μg/mL Pepstatin, 100 μM PMSF, 3.0 μg/mL Aprotinin, 2.5 mM Sodium Pyrophosphate, and 1 mM activated Sodium Orthovanadate in PBS, with a pH of 7.2–7.4.