Computational protocol: Cryopreservation of Primary Mouse Neurons: The Benefit of Neurostore Cryoprotective Medium

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Protocol publication

[…] Neuronal cultures were infected with viruses at DIV 1–2. Mitochondrial superoxide content in neurons was characterized at DIV14 by incubation with 2.5 μM Mitosox Red (Molecular Probes, Invitrogen) for 45 min at 37°C. Next, neurons were fixed in 4% paraformaldehyde and 4% sucrose at room temperature. GFP positive neurons were randomly chosen for quantification. The fluorescence images were acquired in blind using a LSM Zeiss 510 confocal microscope with Zeiss 20×, 40× or 63× objective (Karl Zeiss, Jena, Germany) at a resolution of 2048 × 2048 pixels. All the measurements were performed using NeuronStudio. Neurites were automatically traced and quantified by the software in terms of length, number and morphology (Wearne et al., ; Rodriguez et al., ). Data were then logged and analyzed in Microsoft Excel. For synaptic markers and synapse counts, neurons were similarly fixed at DIV14 and immunostained for MAP2, synapsin1–3 (herein, synapsin) and PSD95. Images were acquired using a Leica SP8-X confocal microscope at 63× at the same resolution. A minimum of 10 neurons per coverslip was imaged. MAP2-positive dendrites were masked using ImageJ (NIH, Bethesda, MA, USA) and synapsin- and PSD95-positive clusters within dendritic masks were quantified using Cell Profiler (Carpenter et al., ). […]

Pipeline specifications

Software tools NeuronStudio, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Caenorhabditis elegans