Cytokines (IFN‐gamma, IL‐10, IL‐12p70, IL‐13, IL‐1ß, IL‐2, IL‐4, IL‐6, IL‐8, TNF‐α, ß‐NGF, and BDNF) from various preparations were analyzed using V‐Plex and U‐Plex human multiplex immunoassay kits on the MSD platform (Meso Scale Diagnostics, Rockville, MD) according to the manufacturer's instructions.
In addition, using Luminex‐based multiplex protein arrays (human 27‐Plex; M500KCAF0Y, BioRad, Hercules CA), the concentrations of SIM and epithelial and endothelial factors were determined. A miniaturized variant of the manufacturer's instructions was used.39 As little as 1‐2 μL of the samples was diluted with sample diluent (1:20) and incubated with multiplex beads for 45 minutes, followed by two washings steps. Afterwards, a cocktail of biotinylated secondary murine antibodies was added for 30 minutes and after final washing steps, the streptavidin‐PE was added. Greater than 50 beads per sample per analyte were detected using the BioPlex Manager 6.2 Software, and concentrations were calculated according to individual standard curves for each analyte ranging from ∼20 ng/mL to the detection limit of ∼2 pg/mL.
In addition, using Luminex‐based multiplex protein arrays (human 27‐Plex; M500KCAF0Y, BioRad, Hercules CA), the concentrations of SIM and epithelial and endothelial factors were determined. A miniaturized variant of the manufacturer's instructions was used.