Cytokine analysis
Cytokine levels in tissue homogenates were measured using the MILLIPLEX Human High Sensitivity T Cell Magnetic Bead Panel HSTCMAG-28SK (Merck Millipore, Darmstadt, Germany), which contained beads for IL-1β, IL-4, IL-5, IL-10, IL-13, GM-CSF, and TNF-α.
The protein concentration of samples was determined using Bradford Protein Assay (Bio-Rad Laboratories, Hercules, United States), according to the manufacturer’s instructions and read by a spectrophotometer (FLUOstar Omega, BMG LabTech, Ortenberg, Germany) at 595nm. The immunoassay was run according to the manufacturer’s protocols. Samples were diluted with assay buffer to a standard total protein concentration of 2mg/mL in a total volume of 50µL. Plates containing standards, controls and samples were read using a validated and calibrated Bio-Plex MAGPIX Multiplex Reader (Bio-Rad, Hercules, United States). Median fluorescent intensity data were analysed using a regression equation for the standard curve to calculate the unknown cytokine concentrations in sample and control wells.