Ferron Sacri R, Andreu-Agullo Celia, Mira Helena, Sanchez Pilar, Marques-Torrejon M Angeles, Farinas Isabel
Departamento de Biología Celular, Universidad de Valencia (UVEG), 46100 Burjassot, Spain.
Nat Protoc. 2007;2(4):849-859. doi: 10.1038/nprot.2007.104.
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.
我们描述了一种由我们团队开发/修改的方案，用于对来自成年小鼠脑室下区（SVZ）的神经干细胞对可溶性因子的反应进行体外和体内评估。该程序包括几个实验选项，这些选项可以单独使用或组合使用。通过神经球培养来测定因子对自我更新、存活和增殖的潜在影响，将因子直接在体外添加到培养板中（步骤1）或在组织解离前立即在体内注入（步骤3）。我们还使用溴脱氧尿苷（BrdU）保留来标记体内缓慢分裂的细胞，随后进行两种不同类型的实验。在一组实验中，将因子添加到从BrdU脉冲动物获得的干细胞原代培养物中，并在免疫细胞化学后测试对标记保留细胞的影响（步骤2）。在另一组实验中，在BrdU脉冲动物中长时间脑室内注入因子后，对完整SVZ中的BrdU标记进行免疫组织化学分析（步骤4）。整个联合程序的最短估计时间为45天。
