Background: Primary cell culture is a valuable tool to utilize in parallel with in vivo studies in order to maximize our understanding of the mechanisms surrounding neurogenesis and central nervous system (CNS) regeneration and plasticity. The zebrafish is an important model for biomedical research and primary neural cells are readily obtainable from their embryonic stages viatissue dissociation. Further, transgenic reporter lines with cell type-specific expression allows for observation of distinct cell populations within the dissociated tissue.
New method: Here, we define an efficient method for ex vivo quantification and characterization of neuronal and glial tissue dissociated from embryonic zebrafish.
Results: Zebrafish brain dissociated cells have been documented to survive in culture for at least 9 days in vitro (div). Anti-HuC/D and anti-Acetylated Tubulin antibodies were used to identify neurons in culture; at 3 div approximately 48% of cells were HuC/D positive and 85% expressed serotonin, suggesting our protocol can efficiently isolate neurons from whole embryonic zebrafish brains. Live time-lapse imaging was also carried out to analyze cell migration in vitro.
Comparison with existing methods: Primary cultures of zebrafish neural cells typically have low rates of survivability in vitro. We have developed a culture system that has long term cell viability, enabling direct analysis of cell-cell and cell-extracellular matrix interactions.
Conclusions: These results demonstrate a practical method for isolating, dissociating and culturing of embryonic zebrafish neural tissue. This approach could further be utilized to better understand zebrafish regeneration in vitro.
Keywords: Cell migration; Primary neuron culture; Time-lapse microscopy; Zebrafish.
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