PI/Calcein-AM double stain kit (Shanghai, China) was used to detect cell apoptosis. After the required treatment, RGCs were incubated with Calcein-AM (10 μM, C3099, Invitrogen) and PI (10 μM, P3566, Molecular Probes) at 37 °C for 20 min. The images were captured using a fluorescence microscope (Olympus IX73, Tokyo, Japan). The live cells were observed at 490 nm excitation filter, while the dead cells were observed at 545 nm excitation filter.

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