Primary cultures of human epithelial corneal cells were established from corneal rims prepared and cultured in flasks as described by Zaidi et al.
31 Cells were seeded at 2 × 10
5 cells per well in 24-well plates and after 18 hours exposed to a 1:20 or 1:10 dilution of the cell-free, sterile supernatant from
S. aureus strains LAC, LACΔPVL, MW2, or MW2ΔPVL grown overnight at 37°C in YCP broth. Aliquots from the wells were collected every 5 minutes and lactate dehydrogenase (LDH) release determined by a commercially available assay (Tox-7 in vitro cytotoxicity assay; Sigma, St. Louis, MO).
The LDH release assay was also used on corneas harvested from infected mice 48 hours postinfection. Female A/J mouse corneas were scratched and infected with 107 CFUs of S. aureus strains LAC, LACΔPVL, MW2, MW2ΔPVL, NRS 193, NRS 193ΔPVL, NRS 194, NRS 194ΔPVL, SF8300, NRS 158, MW2pSO1, MW2pOS1-PVL, NRS 193pOS1, NRS 193pOS1-PVL, NRS 194pOS1, or NRS 194pOS1-PVL for 48 hours. To recover the extracellular LDH present, mouse corneas were washed with 200 μL PBS containing protease inhibitors (Complete Mini; Roche Diagnostics, Mannheim, Germany) and this solution used to determine the LDH activity.