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A combined ex/in vivo assay to detect effects of exogenously added factors in neural stem cells

Nature Protocols volume 2, pages 849–859 (2007)Cite this article

Abstract

We describe a protocol developed/modified by our group for the ex vivo and in vivo assessment of the response to a soluble factor of murine neural stem cells from the adult sub-ventricular zone (SVZ). The procedure includes several experimental options that can be used either independently or in combination. Potential factor effects on self-renewal, survival and proliferation are assayed by means of neurosphere cultures, with the factor administered directly in vitro to the culture plates (Step 1) or infused in vivo immediately before tissue dissociation (Step 3). We also use bromodeoxiuridine (BrdU) retention to label slowly dividing cells in vivo and subsequently perform two different types of experiments. In one set of experiments, the factor is added to primary cultures of stem cells obtained from the BrdU-pulsed animals and effects are tested on label-retaining cells after immunocytochemistry (Step 2). In another set, prolonged intraventricular infusion of the factor in BrdU-pulsed animals is followed by immunohistochemical analysis of BrdU labeling in the intact SVZ (Step 4). The minimum estimated time for the full combined procedure is 45 d.

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Figure 1: The experimental options and timings.
Figure 2: Scheme depicting dissection of the sub-ventricular zone (SVZ; top panel), as explained in Step 1A(ix–xi).
Figure 3: Strategies to label NSCs.

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Acknowledgements

We greatly appreciate the contribution of other lab members to parts of the procedures contained in this protocol. The authors' laboratory is supported by grants from the Spanish Ministerio de Educación y Ciencia and Ministerio de Sanidad y Consumo and from Fundación la Caixa. M.A.M.-T. was a fellow of the Programa de Medicina Regenerativa de la Comunidad Valenciana (MSC y GVA, Spain) and C.A.-A. was a fellow of the MEC-FPU Program. H.M. and P.S. are investigators in the Ramón y Cajal Program (MEC).

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  1. Departamento de Biología Celular, Universidad de Valencia (UVEG), Burjassot, 46100, Spain

    Sacri R Ferrón, Celia Andreu-Agulló, Helena Mira, Pilar Sánchez, M Ángeles Marqués-Torrejón & Isabel Fariñas

  2. Centro de Investigaciones Príncipe Felipe (Unidad Mixta CIPF-UVEG), Valencia, 46013, Spain

    Helena Mira, Pilar Sánchez, M Ángeles Marqués-Torrejón & Isabel Fariñas

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  1. Sacri R Ferrón
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Correspondence to Isabel Fariñas.

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Ferrón, S., Andreu-Agulló, C., Mira, H. et al. A combined ex/in vivo assay to detect effects of exogenously added factors in neural stem cells. Nat Protoc 2, 849–859 (2007). https://doi.org/10.1038/nprot.2007.104

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