Flow cytometry apoptosis measurement

To confirm the effect of NeuroD1 on apoptosis in microglial cells, we detected apoptosis using an Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis kit (Invitrogen, Carlsbad, CA, USA). SH-SY5Y cells were co-cultured in a Transwell plate with BV-2 cells that had been exposed to LPS (1 μg/ml) for 1 h. After LPS stimulation, SH-SY5Y cells were washed and harvested with PBS before being resuspended in 100 μL Annexin-binding buffer containing FITC Annexin V (5 μl) and 100 μg/ml propidium iodide (PI) solution (1 μl) for 15 min in the dark at 22°C. After incubation, 400 μl Annexin-binding buffer was added to each sample before flow cytometry (NOVOCYTE flow cytometer, ACEA Biosciences). Data were analyzed using Novoexpress software (ACEA Biosciences).