Flow Cytometry Quantification of Apoptotic and Necrotic Cell Death

The stratified cultures of control and cytokine exposed human corneal and conjunctival epithelial cells were incubated in the dark at room temperature for 10 minutes in a solution containing 1:2000 propidium iodide and 1:300 FITC-conjugated annexin V (Cayman Chemical, Ann Harbor, MI, USA). The PI positive and FITC negative cells were quantified as necrotic. To calculate apoptotic cells, PI positive FITC negative cells were subtracted from PI positive + FITC negative cell population. The populations of stained cells were analyzed using BD FACSverse flow cytometer (BD Sciences, San Jose, CA, USA). The data were analyzed using FlowJo software.