Chapter 3 96 comet growth speed, kymographs were performed using the Fiji Kymotoolbox plugin (distance, x axis; time, y axis). Live-cell Imaging for Axonal Transport Mitochondrial axonal transport was investigated using previously published protocol (Sousa et al., 2024). Briefly, live imaging of iPSC-derived neurons was performed at DIV39 for co-cultures or DIV2 and 7 for mono-cultures. To analyse mitochondria transport, cultures were incubated at 37°C with medium containing, 25 nM MitoTracker™ Orange CMTMRos (Invitrogen, M7510) for 1 h before live imaging. All moving particles were imaged in a confocal Leica SP8 microscope (Leica Microsystems) with LAS X software (version 3.5.7.23225, RRID:SCR_013673) with HC FLUOTAR L 25x/0.95 W objective. Excitation was carried out using 488 or 561 nm laser lines. Image acquisition was performed for 2 min with 2-2.5 sec frame intervals with a pixel size of 0.116 μm. To analyse axonal transport of moving particles, Kymotoolbox plugin from Fiji/ImageJ was used. The percentage of motile mitochondria was obtained by tracking both motile and stationary mitochondria for 2 min. Neurite Outgrowth and Growth Cone Morphology Neurite outgrowth was addressed in transduced EB3-GFP neurons only, according to previously described protocol (Pinto-Costa et al., 2020). Neuron cultures were fixed with 4% paraformaldehyde (PFA) followed by incubation with mouse anti–βIII-tubulin (1:1,000; Promega, G7121) overnight at 4°C and secondary antibody incubation (donkey anti-mouse– Alexa Fluor 594, 1:1,000; Jackson ImmunoResearch Labs, 715-585-150). Images were acquired by epifluorescence in a Leica DMI6000 B (Leica) equipped with a Hamamatsu FLASH4.0 digital camera and Leica Application Suite Advanced Fluorescence (LAS AF) software (RRID:SCR_013673). Neurite tracing and branching analyses were manually traced using the FIJI NeuronJ plugin. The longest neurite was considered to be the axon. RNA Isolation For RNA isolation from neuron mono-cultures, 1.6M neurons were grown in a density of 40K/cm2. After one week in STEMdiff™ forebrain neuron maturation medium , and feedings on DIV2 and 5, total RNA was isolated using Trizol Reagent (Thermo Fisher Scientific) according to manufacturer's protocol. Samples were sent for bulk mRNA sequencing (NovaSeqXPlus, Illumina technique) to the Core Facility Genomics (Amsterdam UMC, Amsterdam, The Netherlands) and sequenced at a depth of 40M reads per sample.
RkJQdWJsaXNoZXIy MTk4NDMw