We are staining hippocampal neurons with MitoTracker Red and performing time-lapse imaging of mitochondria in axons. But unfortunately, " The percentage of moving mitochondria is considerably less than that in the previous literature, although several mitochondria are visible in axons. How can we increase the percentage of "moving mitochondria" as much as in the previous literature? The staining conditions are 10 min incubation with 25 nM or 10 nM MitoTracker Red. After washing with Neurobasal medium twice, the medium is replaced with Neurobasal+L-Glutamine+NeuroBrew-21 for imaging. In a recent experiment, we have imaged five axons. However, in two axons, we could not observe any moving mitochondria. In the other three axons, we observed 1-3 moving mitochondria in the field of view; however, the other mitochondria did not move at all. In our impression, most moving mitochondria are elongated in morphology. Neurons are imaged every two seconds for 3 min. Time-lapse imaging is stopped within 45 minutes after staining. There is no difference in the percentage of moving mitochondria between DIV2 or DIV3 neurons and DIV7 neurons. We also tried the incubation with 1nM Mitotracker Red for 10 min. In this case, mitochondria look healthy with elongated morphology. However, using our equipment, the MitoTracker signal is too low to perform time-lapse imaging.

Thank you for your help in advance.