A multiplex immunobead assay was us

A multiplex immunobead assay was used to measure the concentrations of interleukin- (IL-) 1, IL-6, tumor necrosis factor- (TNF-) , interferon- (IFN-) , monokine induced by IFN-(MIG), and IFN--induced protein- (IP-) 10 in the conjunctiva, as previously stated.In summary, the conjunctival tissues were collected (four eyes per group) and pooled in lysis buffer containing protease inhibitors for 30 min. The cell extracts were centrifuged at 12,000 rpm for 15 minutes at 4℃, and the supernatants were stored at −70℃until being analyzed. The total protein concentration of the supernatants was determined, and 25 L of total protein of each sample was pipetted into assay plate wells. The supernatants were then added to the wells containing the appropriate cytokine bead mixture that included mouse monoclonal antibodies specific for the cytokines and chemokines for 60 min. After three washes, the plate was incubated for 30 min in the dark at room temperature using a biotinylated detection antibody. The reactions were detected using an analysis system (xPONET, Austin, TX) after the addition of streptavidin-phycoerythrin.The tissue concentrations of the cytokines and chemokines were calculated from the standard curves of known concentrations of recombinant mouse cytokines.