ROS Live Cell Imaging During Neuronal Development.
ROS Live Cell Imaging During Neuronal Development.
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DOI：
10.3791/62165
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发表时间：
2021-02-09
期刊：
影响因子：
--
通讯作者：
Suter DM
中科院分区：
文献类型：
--
作者：
Terzi A;Alam SMS;Suter DM
活性氧（ROS）是公认的信号分子，其在正常发育、稳态和生理学中是重要的。在不同的ROS中，过氧化氢（H2 O2）在细胞信号传导中的作用最具特征。H2 O2在许多物种的发育过程中都有牵连。例如，在受精后的第一天，在斑马鱼胚胎中检测到H2 O2的短暂增加。此外，消耗重要的细胞H2 O2源NADPH氧化酶（NOX）会损害神经系统发育，例如体内和体外视网膜神经节细胞（RGC）的分化、轴突生长和引导。在这里，我们描述了一种方法，用于成像细胞内H2 O2水平在培养的斑马鱼神经元和整个幼虫在发展过程中使用的遗传编码的H2 O2特异性生物传感器，roGFP 2-Orp 1。该探针可以在斑马鱼幼虫中瞬时或稳定表达。此外，比率读数降低了检测到由于差异基因表达或体积效应引起的伪影的概率。首先，我们展示了如何分离和培养来自斑马鱼胚胎的RGCs，瞬时表达roGFP 2-Orp 1。然后，我们使用整个幼虫在组织水平上监测H2 O2水平。该传感器已通过添加H2 O2进行了验证。此外，该方法可用于通过产生具有组织特异性生物传感器表达的转基因动物来测量特定细胞类型和组织中的H2 O2水平。由于斑马鱼促进遗传和发育操作，这里展示的方法可以作为一个管道来测试H2 O2在脊椎动物神经元和一般胚胎发育过程中的作用。该协议描述了使用遗传编码的过氧化氢（H2 O2）-生物传感器在培养的斑马鱼神经元和幼虫的神经系统发育过程中的生理信号作用的评估H2 O2。它可以应用于不同的细胞类型，并通过实验处理进行修改，以研究一般发育中的活性氧（ROS）。
Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the different ROS, hydrogen peroxide (H2O2) is best characterized with respect to roles in cellular signaling. H2O2 has been implicated during development in a number of species. For example, a transient increase in H2O2 has been detected in zebrafish embryos during the first days following fertilization. Furthermore, depleting an important cellular H2O2 source, NADPH oxidase (NOX), impairs nervous system development such as the differentiation, axonal growth and guidance of retinal ganglion cells (RGCs) both in vivo and in vitro. Here, we describe a method for imaging intracellular H2O2 levels in cultured zebrafish neurons and whole larvae during development using the genetically encoded H2O2-specific biosensor, roGFP2-Orp1. This probe can be transiently or stably expressed in zebrafish larvae. Furthermore, the ratiometric readout diminishes the probability of detecting artifacts due to differential gene expression or volume effects. First, we demonstrate how to isolate and culture RGCs derived from zebrafish embryos that transiently express roGFP2-Orp1. Then, we use whole larvae to monitor H2O2 levels at the tissue level. The sensor has been validated by the addition of H2O2. Additionally, this methodology could be used to measure H2O2 levels in specific cell types and tissues by generating transgenic animals with tissue-specific biosensor expression. As zebrafish facilitate genetic and developmental manipulations, the approach demonstrated here could serve as a pipeline to test the role of H2O2 during neuronal and general embryonic development in vertebrates. This protocol describes the use of a genetically encoded hydrogen peroxide (H2O2)-biosensor in cultured zebrafish neurons and larvae for assessing the physiological signaling roles of H2O2 during nervous system development. It can be applied to different cell types and modified with experimental treatments to study reactive oxygen species (ROS) in general development.
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DOI：
10.3791/4196
发表时间：
2012-11-18
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Avdesh A;Chen M;Martin-Iverson MT;Mondal A;Ong D;Rainey-Smith S;Taddei K;Lardelli M;Groth DM;Verdile G;Martins RN
通讯作者：
Martins RN
影响因子：
14.8
作者：
Morgan, Bruce;Van Laer, Koen;Dick, Tobias P.
通讯作者：
Dick, Tobias P.
影响因子：
4.7
作者：
Munnamalai V;Weaver CJ;Weisheit CE;Venkatraman P;Agim ZS;Quinn MT;Suter DM
通讯作者：
Suter DM
影响因子：
16.6
作者：
Ermakova YG;Bilan DS;Matlashov ME;Mishina NM;Markvicheva KN;Subach OM;Subach FV;Bogeski I;Hoth M;Enikolopov G;Belousov VV
通讯作者：
Belousov VV
影响因子：
4.8
作者：
Oparka, Monika;Walczak, Jaroslaw;Wieckowski, Mariusz R.
通讯作者：
Wieckowski, Mariusz R.