Brainphys imaging optimized medium

For confocal imaging, the monocytes were labeled with Vybrant DiL (red) (Invitrogen) at 1:1000 dilutions at 37 °C for 30 min before seeding onto hCOs. A total of 1 × 10⁵ cells of labeled monocytes were added to each hCO of 6 weeks old with 2 mL of BrainPhys Imaging Optimized medium (Stemcell Technologies). After coculturing in a 37 °C incubator for 65 h, the hCOs were stained with Cal520 calcium dye (Abcam) at 1:1000 dilutions at 37 °C for 30 min and then washed twice with 1× PBS. Pictures of neuron damage were taken using an Olympus OSR spinning disk microscope at 60× oil objective lens.

A Fluo-4 Direct calcium assay kit (Invitrogen) was used according to the manufacturer’s instructions to determine calcium activities. In short, explants on culture inserts (0.4 μm Millicell, Merck Millipore) were incubated with a mixture of BrainPhys™ Imaging Optimized Medium (Stemcell Technologies) and Fluo-4 calcium imaging reagents (1:1 v/v) for 1 h at 37 °C. After incubation, the brain explants were harvested by cutting the semi-permeable membrane from the culture insert and placing it upside down on imaging dishes (Ibidi). Spontaneous calcium fluctuations were recorded at 37 °C by fluorescence confocal microscopy (Leica SP5) at 1 frame per 1.29 s.

To measure mitochondrial neurite transport, iPSC-derived sMNs at differentiation day 30 were incubated with 50 nM MitoTracker™ Green FM (M7514, Invitrogen) in neuronal medium for 20 min at 37 ℃. After 20 min, cells were washed and incubated in BrainPhys™ Imaging Optimized Medium (05796, STEMCELL Technologies) for imaging. Images were taken using the Operetta CLS High-Content Analysis System (PerkinElmer) with a 40 × objective at 37 ℃ and 5% CO₂. The MitoTracker™ Green was excited at ~495 nm and 1-s time-lapse images were taken for 200 s. Video files were analyzed with ImageJ using TrackMate v3.8.0 plugin for total mitochondria quantification and tracking, and time/distance kymographs to quantify the number of moving mitochondria in a selected neurite segment. Moving mitochondria are represented by tilted lines, whereas stationary mitochondria can be discerned as straight vertical lines.