The level of extracellular ROS prod

The level of extracellular ROS production was measured using a DCFDA kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol, as previously mentioned . In summary, surgically excised mice conjunctiva (four eyes per group) was washed with PBS and 10 M DCFDA and then incubated at 37℃ for 20 minutes. To determine the extracellular ROS production, the cell pellet was analyzed using a FACSCalibur flow cytometer at excitation and emission wavelengths of 488 and 530 nm, respectively. The data analysis was based on 10,000 detected events using flow and image cytometry analysis software. The relative changes in DCFDA fluorescence were expressed as the fold increase compared to that in the UT group.