To measure mitochondrial size, Prox1::eGFP and DsRed2-mito were co-expressed in DG-like neurons via lentiviral infection. Neurons were fixed in 4% paraformaldehyde and then permeabilized with 0.1% Triton-X100 in TBS. Cells were then blocked in TBS containing 3% donkey serum for 1 h, followed by incubation with DAPI for 15 min. Fluorescence images were acquired using a high-resolution LSM 710 confocal microscope (Carl Zeiss) and were processed with ZEN 2011 software (Carl Zeiss) and Adobe Photoshop CS5 software (Adobe). The size of the mitochondria (DsRed2-mito puncta) was analysed using the Particle Analysis tool in ImageJ software (National Institutes of Health).
For MMP, neurons were incubated with JC-1 dye (Molecular Probes) at 37 °C for 15–30 min29 (link). The cells were dissociated into single cells using TrypLE (Invitrogen), washed three times and then resuspended in 1 ml warm PBS. Green and red fluorescence of JC-1 dye was quantitated using BD FACSCanto II flow cytometer (Becton, Dickinson). Histogram plots of green and red fluorescence were created to determine the red/green intensity ratio using FlowJo 10 software (TreeStar).
For MMP, neurons were incubated with JC-1 dye (Molecular Probes) at 37 °C for 15–30 min29 (link). The cells were dissociated into single cells using TrypLE (Invitrogen), washed three times and then resuspended in 1 ml warm PBS. Green and red fluorescence of JC-1 dye was quantitated using BD FACSCanto II flow cytometer (Becton, Dickinson). Histogram plots of green and red fluorescence were created to determine the red/green intensity ratio using FlowJo 10 software (TreeStar).
