AO/EB Staining for Cell Viability

Viability of the IECs treated with or without H₂O₂ was evaluated by double staining with AO/EB (Sigma). After removing the H₂O₂, the cells were washed with PBS and trypsinized. A mixture containing 100 µL of the cell suspension containing about 10⁵ cells/mL in PBS and 10 µL of the AO/EB working solution (50 µg/mL of AO and 50 µg/mL of EB in PBS) was then prepared. The cells were immediately analyzed with a fluorescence microscope (Olympus IX71; Olympus Corporation). Cell death rate was monitored by trypan blue exclusion with a hemocytometer (Nexcelom Bioscience, USA) as previously described [19 (link)].