Name: optical quantification of intracellular ph in drosophila melanogaster malpighian tubule epithelia with a fluorescent genetically-encoded ph indicator
Uploaded: 2017-08-11T12:30:00
Description: Watch this Scientific Journal Video about Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator at JoVE.

这项工作得到了 NIH DK092408 和 DK100227 对生产商的支持. AJR 得到了 T32-DK007013 的支持。作者希望感谢 Dr. 科尔尼 CapaR-GAL4 和 c724-GAL4 的果蝇股票。我们还感谢雅各布 b. 安德森协助维持实验飞行十字架。

本协议中的所有步骤均符合梅奥诊所 (罗切斯特, 锰) 动物使用指南。
1. 畜牧业
<ol><li>饲养苍蝇和设置十字架根据标准畜牧业29。 注: 荧光报告器的 GAL4/UAS 系统的表达与温度成正比, 从而可以调节培养温度以改变表达水平。虽然高表达式水平往往会导致更好的信噪比这一条件也与增加胞浆和 organellar 聚合当使用 GFP 的红色荧光蛋白 (RFP) 融合结构, 如 pHerry<sup...

在果蝇mts 中, pH 值i的量化成功与否完全取决于提取的 mts 的运行状况以及安装和剥离的质量 (图 A - C)。因此, 仔细处理组织的描述是势在必行的。新涂在 PLL 上的幻灯片实质上有助于 MT 的安装, 因为它们比以前暴露在溶液中的幻灯片更容易粘合。小心安装也有助于识别不同的 MT 段 (图 D)。健康 MTs 分别通过减少 mCherry 聚合和产生更?...

本议定书的目的是用基因编码的 ph 指示剂 (GEpHI) 描述细胞内 ph 值 (ph 值i) 的量化, 并演示这种方法如何用于评估模型昆虫中的侧 H+传输 (D。腹氏管 (MT) 的肾脏结构。MTs 作为果蝇的排泄器官, 在几个关键方面与哺乳动物肾单位的功能相似,1。在苍蝇的胸腔和腹部, MTs 排列为2对小管 (前后)。每个 MT 的单细胞上皮管由新陈代谢活性主细胞组成, 具有明显的顶端 (腔) 和侧 (腔) 极性, 以及夹层星状细胞。前 MTs 由3形态、功能和发育的不同部分组成, 特别是最初的扩张段、移行段和分泌主段, 它们连接到输尿管的2。在细胞尺度的跨上皮离子传输到流明是由一个顶端的等离子膜 V atp 酶3和一个碱金属/H+换热器以及一个侧 Na+-K-atp 酶4,内整流 K+通道5, Na+驱动 Cl−/小贩3−交换器 (NDAE1)6和 Na+-K+2Cl− cotransporter (NKCC;Ncc69)7, 而星状细胞介导 Cl-和水传输8,9。这一复杂但容易接近的生理系统提供了极好的机会, 研究内源性离子转运机制时, 结合了不同的遗传和行为工具的果蝇。
这个协议的基本原理是描述一个遗传可塑性的系统研究上皮离子运输有潜在的集成从细胞到行为和出口的工具到其他模型系统。pHerry10的表达式, GEpHI 从绿色 ph 值敏感的 super-ecliptic pHluorin11、12 (SEpH) 和红色 ph 不敏感的 mCherry13的融合中派生而来, 在 MTs 中允许量化 H+传输单 MT 细胞通过高 K+/尼日利亚校准技术14。由于许多离子转运体移动了 H+当量, 细胞内 pH 值i的量化是通过各种转运体的离子运动的功能表征。果蝇MT 模型系统还提供了功能强大的基因工具在组织特异转基因15和 rna 干涉 (干扰)16表达式, 可以结合细胞成像和全身检测17,18,19的小管函数创建一个具有从分子到行为的垂直集成的健壮工具集。这与许多其他用于评估上皮生物学的协议相对照, 因为历史上这种测量依赖于复杂而艰巨的微解剖, 尖端的离子选择性电极20,21,和昂贵的 pH 敏感染料22 , 具有限制性的负载要求和不均匀组织中的细胞特异性差。GEpHIs 已被用来广泛测量 pHi在各种细胞类型23。早期工作利用绿色荧光蛋白 (GFP) 的固有 ph 敏感度来监测培养的上皮细胞中的 ph 值i 24但过去20年中, 在神经元中使用过 GEpHIs25、胶质26、真菌27和植物细胞28。通过 GAL4/UAS 表达系统15和果蝇MT 的生理可访问性, 将基因构造的细胞靶向性结合起来, 使之成为研究 pH 值的理想准备.调节和上皮离子转运。
pH 值i规则已进行了几十年的研究, 对生命至关重要。MT 制剂提供了一个健壮的模型来教授生理学的 ph 值i调节, 但也执行复杂的研究 ph 值i调节ex 体内和体内。本协议描述了使用 NH4Cl 脉冲酸加载技术21, 在果蝇MT 的上皮细胞的侧膜上进行 H+运动的量化, 但由于 pH 值是基因编码, 这些方法和他们的理论框架可以适用于任何准备转基因和活成像。

<table>
	<tbody>
		<tr>
			<td>Poly-L-Lysine Solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4832</td>
			<td>Store at 4 &#176;C, can be reused.</td>
		</tr>
		<tr>
			<td>Nigericin Sodium Salt</td>
			<td>Sigma-Aldrich</td>
			<td>N7143</td>
			<td>CAUTION: Handle with gloves. Store as aliquots of 20 mM stock solution in DMSO at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Adhesive Perfusion Chamber Covers, adhesive size 1 mm, chamber diameter &#215; thickness 9 mm &#215; 0.9 mm, ports diameter 1.5 mm</td>
			<td>Sigma-Aldrich</td>
			<td>GBL622105</td>
			<td>Can be substituted as needed to match perfusion system.</td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Ellsworth Adhesives</td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>Helping Hands Soldering Stands</td>
			<td>Harbor Freight Tools</td>
			<td>60501</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>Open Gravity-fed Perfusion System with Valve Controller, 8 to 1 Manifold and Reserviors</td>
			<td>Bioscience Tools</td>
			<td>PS-8S</td>
			<td>Any comparable perfusion system can be used.</td>
		</tr>
		<tr>
			<td>Flow Regulator</td>
			<td>Warner Instruments</td>
			<td>64-0221</td>
			<td>Can be substituted as needed to match perfusion system.</td>
		</tr>
		<tr>
			<td>Schneider&#39;s Medium</td>
			<td>Fisher Scientific</td>
			<td>21720024</td>
			<td>Store at 4 &#176;C in sterile aliquots.</td>
		</tr>
		<tr>
			<td>#5 Inox Steel Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td>Can be substituted based on experimenter comfort.</td>
		</tr>
		<tr>
			<td>35 mm x 10 mm polystyrene Petri dish</td>
			<td>Corning Life Sciences</td>
			<td>Fisher Scientific 08-757-100A</td>
			<td>Exact brand and size are unimportant.</td>
		</tr>
		<tr>
			<td>75 x 25 mm Microscope Slides</td>
			<td>Corning Life Sciences</td>
			<td>2949-75X25</td>
			<td>Exact brand and size can vary as long as perfusion wells are compatible.</td>
		</tr>
		<tr>
			<td>Filimented Borosilicate Capillary Glass, ID 1.5 mm, OD 0.86 mm, thickness 0.32 mm</td>
			<td>Warner Instruments</td>
			<td>64-0796</td>
			<td>Filiment not necessary, glass can be substituted to match perfusion tubing and perfusion wells.</td>
		</tr>
		<tr>
			<td>Tygon Tubing, ID 1/16 inch, OD 1/8 inch, thickness 1/32 inch</td>
			<td>Fisher Scientific</td>
			<td>14-171-129</td>
			<td>Available from multiple vendors, can be substituted to match perfusion system.</td>
		</tr>
		<tr>
			<td>Vacuum Silicone Grease</td>
			<td>Sigma-Aldrich</td>
			<td>Z273554</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>Plastic Flow Control Clamp</td>
			<td>Fisher Scientific</td>
			<td>05-869</td>
			<td>Available from multiple vendors, sterility not required</td>
		</tr>
		<tr>
			<td>Glass rods, 5 mm diameter</td>
			<td>delphiglass.com</td>
			<td>9198</td>
			<td>Exact size is personal preference, multiple vendors available</td>
		</tr>
		<tr>
			<td>PAP Hydrophobic Pen</td>
			<td>Sigma-Aldrich</td>
			<td>Z377821</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>Sealing Film</td>
			<td>Sigma-Aldrich</td>
			<td>P7668</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>15 mL Falcon tube</td>
			<td>BD Falcon</td>
			<td>352096</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>50 mL Falcon tube</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>HEPES; 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>MES; 4-Morpholineethanesulfonic acid monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>69892</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>TAPS; N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T5130</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>10x/0.45 Air Objective</td>
			<td>Zeiss</td>
			<td>000000-1063-139</td>
			<td>Comparable objectives can be substituted. 40x objectives can be used for single cell imaging.</td>
		</tr>
		<tr>
			<td>Dissecting Stereoscope</td>
			<td>Zeiss</td>
			<td>Discovery.V8</td>
			<td>Any dissecting stereoscope can be used.</td>
		</tr>
		<tr>
			<td>UAS-pHerry transgenic Drosophila melagnogaster</td>
			<td>Available from Romero Lab</td>
			<td></td>
			<td>First published: Citation 10</td>
		</tr>
		<tr>
			<td>capaR-GAL4 driver line Drosophila melagnogaster</td>
			<td>Available from Romero Lab</td>
			<td></td>
			<td>First published: Citation 32</td>
		</tr>
		<tr>
			<td>c724-GAL4 driver line Drosophila melagnogaster</td>
			<td>Available from Romero Lab</td>
			<td></td>
			<td>First published: Citation 2</td>
		</tr>
		<tr>
			<td>Monochromatic High Sensitivity Digital Camera</td>
			<td>Zeiss</td>
			<td>Axiocam 506 mono</td>
			<td>Exact brand and model can vary, can be replaced with any monochromatic high-sensitivity camera suited to live cellular imaging.</td>
		</tr>
		<tr>
			<td>GFP/FITC filter set, 470/40 nm ex., 515 nm longpass em., 500 nm dichroic</td>
			<td>Chroma</td>
			<td>CZ909</td>
			<td>Any GFP/FITC filer set can be substituted.</td>
		</tr>
		<tr>
			<td>RFP/TRITC filter set, 546/10 nm ex., 590 nm longpass em., 565 nm dichroic</td>
			<td>Chroma</td>
			<td>CZ915</td>
			<td>Any GFP/FITC filer set can be substituted.</td>
		</tr>
		<tr>
			<td>Inverted Epifluoescent Microscope</td>
			<td>Zeiss</td>
			<td>Axio Observer Z.1</td>
			<td>Any comparable microscope with motorized filter switching can be used. Upright microscopes can be used with open perfusion baths and water-immersion objectives.</td>
		</tr>
		<tr>
			<td>Statistical Analysis Software</td>
			<td>Microcal</td>
			<td>Origin 6.0</td>
			<td>Any software with comparable functionality can be substituted</td>
		</tr>
		<tr>
			<td>Image Analysis Software</td>
			<td>National Institutes of Health</td>
			<td>ImageJ 1.50i</td>
			<td>Any software with comparable functionality can be substituted</td>
		</tr>
		<tr>
			<td>Image Acquisition Software</td>
			<td>Zeiss</td>
			<td>Zen 1.1.2.0</td>
			<td>Any software with comparable functionality can be substituted</td>
		</tr>
		<tr>
			<td>Single-edged Carbon Steel Razor Blade</td>
			<td>Electron Microscopy Sciences</td>
			<td>71960</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>Microscopy Slide Folder</td>
			<td>Fisher Scientific</td>
			<td>16-04</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>Bunsen Burner</td>
			<td>Fisher Scientific</td>
			<td>50-110-1231</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>Polystrene Drosophila Rearing Vials with Flugs</td>
			<td>Genesee Scientific</td>
			<td>32-109BF</td>
			<td>Comparable items can be substituted.</td>
		</tr>
		<tr>
			<td>2.5 L Laboratory Ice Bucket</td>
			<td>Fisher Scientific</td>
			<td>07-210-129</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>NMDG; N-Methyl-D-glucamine</td>
			<td>Sigma-Aldrich</td>
			<td>M2004</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>200 uL barrier pipette tips</td>
			<td>MidSci</td>
			<td>AV200</td>
			<td>Available from multiple vendors.</td>
		</tr>
		<tr>
			<td>200 uL variable volume pipette</td>
			<td>Gilson Incorporated</td>
			<td>PIPETMAN P200</td>
			<td>Available from multiple vendors.</td>
		</tr>
	</tbody>
</table>

optical quantification of intracellular ph in drosophila melanogaster malpighian tubule epithelia with a fluorescent genetically-encoded ph indicator

上皮离子转运对系统离子稳态和基本细胞电化学梯度的维持至关重要。细胞内 ph 值 (ph 值i) 受许多离子转运体的影响, 因此监测 ph 值i是评估转运体活动的有用工具。现代基因编码的 ph 指标 (GEpHIs) 提供了在细胞和亚细胞尺度上的完整单元的 ph 值i的光学定量。本议定书描述 real-time 定量的细胞 pHi调节在氏管 (MTs) 的果蝇通过ex 活体成像 pHerry, pseudo-ratiometric GEpHI 与 pK非常适合跟踪胞中的 pH 值变化。提取的成人蝇 MTs 是由形态和功能分明的单细胞层上皮的部分组成, 并可作为一个容易接近的和基因驯服的研究上皮运输模型。GEpHIs 提供了一些优于传统 pH 敏感荧光染料和离子选择电极的优势。GEpHIs 可以标记不同的细胞数量提供适当的启动子元素可用。此标记在ex 体内、体内和原位准备工作中特别有用, 它们本质上是异构的。GEpHIs 也允许定量的 pH 值i在完整的组织, 而无需重复染料治疗或组织外化。当前 GEpHIs 的主要缺点是在组织损伤和构造表达的反应中, 聚集在胞浆包裹体中的倾向。这些缺点, 它们的解决方案, 和 GEpHIs 的内在优势, 通过评估侧质子 (H) 在功能分明的主要和星状细胞中提取的飞行 MTs 的传输来证明.所描述的技术和分析可以很容易地适应各种各样的脊椎动物和无脊椎动物的准备工作, 而且化验的复杂程度可从教学实验室扩展到通过特定转运体对离子通量的复杂测定。

健康的组织和正确的识别前 MTs 是至关重要的成功的协议。在解剖过程中, 应注意不要直接接触 mts, 而只能由输尿管来处理, 因为握住 mts 直接会导致破损 (图 4A- B)。当 MTs 被平扫到滑梯上时, 小管必须尽可能少地接触, 避免多余的运动, 因为这会损害单细胞上皮层 (图 4C)。正确解剖前 MTs, 通过胞上皮细胞?...

Watch this Scientific Journal Video about Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator at JoVE.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

细胞离子传输通常可以通过监测细胞内 ph 值(ph 值i)来评估。基因编码的 ph 值指标(GEpHIs)提供了细胞内 ph 值的光学定量。本议定书详细介绍了细胞内 ph 值的量化, 通过蜂窝状的前体活成像的氏管的果蝇与 pHerry, pseudo-ratiometric 基因编码的 pH 指标。

用荧光基因编码的 ph 指示剂对果蝇氏小管上皮细胞内 ph 值的光学定量

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Research

JoVE Journal

Biology

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Dissection of Larval CNS in Drosophila Melanogaster

在果蝇幼虫中枢神经系统的剖析

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

科研

生物学

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

在幼虫的监测心脏功能果蝇生理研究

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Measurement of Lifespan in Drosophila melanogaster

Measurement of Lifespan in Drosophila melanogaster

寿命测量<em&gt;果蝇</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

液泡和胞质pH值的测量<em&gt;在体内</em在酵母细胞悬浮液

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

基因编码的分子探针来研究G蛋白偶联受体

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

使用拉姆齐试验来测量液体分泌和离子流率在<em&gt;果蝇</em&gt;马氏管

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The ...

Maintenance of a Drosophila melanogaster Population Cage

Maintenance of a Drosophila melanogaster Population Cage

的维持<em&gt;果蝇</em&gt;人口笼

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Application of Genetically Encoded Fluorescent Nitric Oxide NOÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬Ãƒâ€šÃ‚Â¢ Probes, the geNOps, for Real-time Imaging of NOÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬Ãƒâ€šÃ‚Â¢ Signals in Single Cells

遗传编码的荧光一氧化氮的应用（NO•）探针，所述geNOps，用于在单细胞NO•信号的实时影像

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

果蝇成人间接飞行肌的光学横截肌面积测定

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight ...