Isolation and Characterization of Murine Conjunctiva-Associated Lymphoid Tissue

The eyes and lids of mice (n=5 per experiment, in four independent sets of experiments, total of 20 per group, in NS, DS5, and DS10 groups and n=5 per experiment, in two independent sets of experiments in corneal scar and control groups) were excised, pooled, and incubated in 10ml of 5mgml⁻¹ Dispase II (Roche Molecular Biochemicals, Indianapolis, IN) in a shaker at 37 ° C for 1 h, followed by neutralization with Hank ’s Buffered Salt Solution (Invitrogen-Gibco, Grand Island, NY) supplemented with 3% fetal bovine serum (Hyclone, Logan, UT). The bulbar and tarsal conjunctivae were scraped with cytology brushes under a dissecting microscope. Respective superficial CLNs were surgically excised, smashed in between two sterile frosted glass slides, and made into a single-cell suspension. Cell populations were individually collected, centrifuged at 2000r.p.m.×5min, filtered, and resuspended. Cells collected by this technique were used either for flow cytometry (desiccating stress experiment) or for ELISPOT (desiccating stress and corneal ulcer experiment).