近质膜和全球星形胶质细胞内钙动力学测量
Summary
我们描述了如何来衡量膜和全球培养的星形胶质细胞，利用全内反射和epifluorescence显微镜细胞内钙离子动力学附近。
Abstract
大脑含有神经胶质细胞。星形胶质细胞，神经胶质细胞类型，长期被称为神经元提供了一个被动的支撑作用。然而，越来越多的证据表明，星形胶质细胞可能也积极参与脑功能，通过与神经元的功能的相互作用。然而，星形胶质细胞生物学的许多基本方面仍然争议，职责不清和/或实验未开发。其中一个重要的问题是星形胶质细胞内钙瞬变的动态。这是有关的，因为钙是完善作为一项重要的第二信使，因为它已被提出，星形胶质细胞钙升高可以触发从星形胶质细胞释放发射机。然而，还没有任何近质膜钙信号在星形胶质细胞的详细或令人满意的描述。全内反射荧光显微镜（TIRF）是一个强大的工具来分析有关生理信号事件，约100纳米的活细胞质膜内。在这里，我们使用TIRF显微镜和描述如何几乎同时监测到附近的细胞膜和全球细胞内钙的动态。这种方法的进一步完善和系统应用的潜力，以了解星形胶质细胞钙信号的精确细节。一个星形胶质细胞钙动力学的详细了解可能会提供一个基础，了解是否，如何，何时和为什么星形胶质细胞和神经元接受钙依赖性的功能上的相互作用。
Protocol
实验步骤实验过程由两个关键部件，是明智的方式在一个步骤如下所述。 第1部分：准备海马星形胶质细胞的培养简单地说，混合海马星形胶质细胞神经元的文化准备使用一个完善的协议1,2,3。我们优化的过程，产生健康的培养的星形胶质细胞。下面列出的所有程序应在无菌层流罩，如环境进行。 准备…
Discussion
它是行之有效的，星形胶质细胞显示细胞内钙升高。这些自发地发生，可引发神经元的活动，或通过受体激动剂的应用程序激活星形胶质细胞表面的受体11。其中一个重要的和有争议的问题是是否星形胶质细胞内钙升高可触发释放信号分子，激活受体对神经元的11，12。因为有支持和反对这种观点的证据， 海顿7，第13和麦卡锡 11实验室在评论强调，这是有争议的。根据我们最近的?…
Acknowledgements
这项工作是支持日本的上原纪念基金会（ES）以及白厅基金会，国立神经疾病与中风和斯坦 – 奥本海默基金会奖（BSK）。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| VWR® Micro Cover Slips, Round, No. 1 | Tool | VWR | 48380-068 | |
| Poly-D-lysine hydrobromide | Reagent | Sigma | P0899 | |
| Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane | Reagent | Sigma | L2020 | |
| Earle’s Balanced Salt Solution (EBSS) (1X), liquid | Reagent | Invitrogen | 14155-063 | |
| Minimum Essential Medium (MEM) (1X), liquid Contains Earle’s salts, but no L-glutamine or phenol red | Reagent | Invitrogen | 51200-038 | |
| Penicillin-Streptomycin liquid | Reagent | Invitrogen | 15140-122 | |
| Sodium pyruvate solution | Reagent | Sigma | S8636 | |
| HEPES solution 1 M | Reagent | Sigma | H0887 | |
| N-2 Supplement (100X), liquid | Reagent | Invitrogen | 17502-048 | |
| Horse Serum, Heat-Inactivated | Reagent | Invitrogen | 26050-088 | |
| PAPAIN-022 | Reagent | Worthington | LK003178 | |
| Neurobasal™ Medium (1X) Liquid without Phenol Red | Reagent | Invitrogen | 12348-017 | |
| B-27 Serum-Free Supplement (50X), liquid | Reagent | Invitrogen | 17504-044 | |
| L-Glutamine-200 mM (100X), liquid | Reagent | Invitrogen | 25030-149 | |
| Cell Strainers | Tool | BD Biosciences | 352350 | |
| BD Falcon Multiwell Flat-Bottom Plates with Lids, Sterile | Tool | BD Biosciences | 353046 | |
| NaCl | Reagent | Sigma | S7653 | |
| KCl | Reagent | Sigma | P3911 | |
| CaCl2 hexahydrate | Reagent | Sigma | 21108 | |
| MgCl2 hexahydrate | Reagent | Sigma | M2670 | |
| HEPES free acid | Reagent | Sigma | H3375 | |
| D-(+)-glucose | Reagent | Sigma | G7528 | |
| Fluo-4, AM 1 mM solution in DMSO | Reagent | Invitrogen | F-14217 | |
| Pluronic® F-127 20% solution in DMSO | Reagent | Invitrogen | P-3000MP | |
| Immersion Oil TYPE DF | Microscope | Cargille | 16242 | |
| Open chamber for 25 mm round coverslips, 100 μl volume | Tool | WARNER Instruments | 64-0362 (RC-21BDW) | |
| P-2 platform for Series 20 chambers, non-heater | Tool | WARNER Instruments | 64-0278 (P-2) | |
| FluoSpheres carboxylate-modified microspheres, 0.1 μm, yellow-green fluorescent (505/515) 2% solids | Reagent | Invitrogen | F8803 |
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Cite This Article
Shigetomi, E., Khakh, B. S. Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes. J. Vis. Exp. (26), e1142, doi:10.3791/1142 (2009).