2.7. Calcein-AM, and Propidium Iodide (PI) Staining

Viable or dead cells were stained with Calcein-AM or PI as previously described [5]. After treating neurons with glutamate (20 μM in sterile PBS), Calcein-AM (25 nM in DMSO), PI (0.5 μM in sterile PBS), or Hoechst (1 μg/mL in sterile PBS) (Thermo Fisher Scientific, Waltham, MA, USA) was added into the culture medium for 30 min at 37 °C in the dark. Micrographs were taken using a Zeiss Axiovert A1 microscope (Zeiss, Oberkochen, Germany) using a consistent exposure time. The number of PI-positive neurons, Hoechst positive neurons, or calcein fluorescence density per cell was analyzed using AxioVision 4.9.