Visualizing Mitochondrial Dynamics in HL-1 Cells

Mitochondria were visualized using CellLight™ Mitochondria-GFP, BacMam 2.0 (ThermoFisher Scientific, Waltham, MA, USA). To visualize mitochondrial movement in living cells, the HL-1 cells were cultured in NuncTM Glass Base Dish, 12 mm (ThermoFisher Scientific, Waltham, MA, USA). The HL-1 cells were incubated with a studied factor for 1 h (5 µM iFBP2, 10 µM GSK3 inhibitor SB216763, 1 µM PI3K inhibitor wortmannin, 5.5 mm glucose; Merck, Darmstadt, Germany) and images were acquired using the Time Scan option the FV-1000 confocal microscope. Time-lapse videos were created on 3× digital zoom and analyzed using FV-10-ASW 4.2 Viewer software (Olympus). To avoid the measurement of random movement of mitochondria powered by cytoplasm fluctuations only the organelles that moved at least 2 μm during the image acquisition time were taken into account [22 (link)]. If a mitochondrion moved with pausing episodes, the mean velocity was counted rejecting pause time. The experiments were replicated at least three times, and velocities of at least 482 mitochondria were measured for each treatment.
The length and shape of mitochondria were analyzed using the MiNA (Mitochondrial Network Analysis by Sturat Lab) plugin of the ImageJ/FIJI. At least 5480 mitochondria were measured in each condition.