Mitochondrial live-cell imaging

Neurospheres were plated onto glass bottom 35 mm dishes. Before imaging, cells were treated with 25 nM MitoTracker Red CMXRos (Invitrogen). Then, cells were washed three times with imaging medium, and the dish was placed into a live-cell imaging chamber at 37°C and 5% CO₂. Axons were identified using morphological characteristics (i.e. constant thin diameter, long neurites, no proximal branching, and direct emergence from the cell body), followed by live-cell imaging as previously described.^34,35 Briefly, images of mitochondria along axons were acquired every 5 s for a total of 5 min. Quantitation of the movement of each mitochondrion was performed using ImageJ software by an analyser who was blind to experimental conditions. After tracing each mitochondrion, kymographs of mitochondrial movement were generated (x-axis represents position along axons and y-axis represents time). A minimum of 120 mitochondria from three independent experiments were analysed per group. The velocity of mitochondria showing any movement at the anterograde or retrograde direction³⁶ was examined. Since mitochondria move more frequently in the anterograde direction in our cultures, the number of anterograde mitochondria is more than that in the retrograde direction. Motile mitochondria are defined as moving more than ∼5 µm forward or backward from the origin during the recording period (≥0.016 µm/s).^35,37 Multiple transport parameters including average velocity of mitochondria, the percentage of motile mitochondria, and the frequency of motile mitochondria in both anterograde and retrograde directions were calculated and compared between groups.