Non-stimulated tear fluids were collected from the tear lake inside the lateral conjunctival sac of the inferior fornix with microcapillary tubes (5 μL, Drummond Scientific, Broomall, PA), dispensed into an Eppendorf microtube (Protein LoBind, VWR, Batavia, IL), and frozen immediately at −70°C. At the end of the study, all tear samples were analyzed for concentrations of 43 protein analytes using microsphere-based immunoassay (Luminex, Austin, TX) in 3 multiplex panels and lysozyme assay. Briefly, microtubes first were centrifuged and tear fluid of precise volume from each sample was transferred to a new tube containing PBS sample buffer (with 4% BSA, 20% fetal calf serum [FCS], and 0.01% SDS) and undergoing analysis using standardized assay procedures. Final dilution factor was 1:12 for 2 multiplexes of cytokines, 1:25,000 for Ig panel and 1:1,000,000 for lysozyme simplex. For tear samples with <3 μL but at least 1 μL available, higher dilution (up to 36-fold for cytokine panels) was used. Tear samples with <1 μL tear fluid available were excluded from analysis. The concentration of each protein analyte was determined from standard curves.