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.