Measurement of Goblet Cell Survival

Conjunctival goblet cell survival was determined with a lactate dehydrogenase (LDH) assay (MAK401; Sigma-Aldrich, Shiga, Japan) used after treatment of cells. This assay measures the amount of LDH, a cytoplasmic enzyme, release from the cells. Goblet cells were exposed to BT, DS, OP, or LT diluted 1:7 in medium (volume 200 μL) for 30 min or 6 h in an incubator at 37°C and 5% CO₂. As the control, cells were treated with basal culture medium. After the treatment, the medium was changed, and the cells were left in the fresh medium for an additional 20 h before measurement of LDH release. The medium was removed and centrifuged at 500 rpm for 10 min. Each supernatant was transferred to a corresponding well in a new plate. To measure the amount of LDH remaining in the cells, 200 μL of 1% Triton-X (1001325622; Sigma-Aldrich, St. Louis, Missouri, USA) was added to each well in the original plate and incubated for 10 min at room temperature (RT). Plates were centrifuged, and each supernatant was transferred to a corresponding well. LDH solution was added and incubated for 3–15 min at RT; 1M HCL was added to each well, bubbles were removed, and the plates were read at 490 nm on the SpectraMax i3X multi-mode microplate reader (Molecular Devices, San Jose, CA, USA). Cell survival was assessed as the ratio between LDH release prior to membrane permeation by Triton-X and total LDH. Percentage compared to control was calculated. Cell survival was analyzed on at least 3 cell cultures from different donors.