Multimodal Imaging of Mitochondria and Lysosomes

Mitochondria were stained with MitoTracker^® Red CMXRos (M7512 ThermoFisher, MT-R). Acidic organelles were stained by Lysotracker Green DND-26 (#8783 Cellsignal, LT-G). For Mitotracker and Lysotracker stainings, cells were incubated for 30 min with Mitotracker (50 nM) and Lysotracker (50 nM) in prewarmed, freshly prepared culture medium (50% total medium change). In MN grown in microfluidic chambers, mitochondria were labeled by addition of a lentivirus overexpressing MitoDsRed (Prots et al., 2018 (link)). To analyze mitochondrial membrane potential, JC-1 (T3168, Invitrogen) was added to the medium at a final concentration of 1 μg/ml (50% medium change to prevent detachment) and cells were recorded after 60 min. As a positive control of mitochondrial depolarization, CCCP (50 μM final concentration, diluted in ethanol) was added to the medium 30 min before imaging. Lysosomes were labeled using immunohistochemistry for Lamp1 (see below) on day 31 differentiated neuronal cultures. In the ImageJ/Fiji software, the Lamp1 channel was thresholded using the auto function and the area of Lamp1 was determined within the region of the cell soma. For the quantification of Lamp1 positive organelles (LPO), enlarged perinuclear Lamp1 organelles were manually counted on blindcoded slides.