Name: use of the ramsay assay to measure fluid secretion and ion flux rates in the drosophila melanogaster malpighian tubule
Uploaded: 2015-11-25T15:00:00.000+00:00
Duration: 13 min 30 s
Description: Watch this Scientific Journal Video about Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule at JoVE.com

The authors wish to thank Drs. Sung-wan An and Mike O&#8217;Donnell for practical advice on establishing this assay, Dr. Chih-Jen Cheng for helpful discussions on the use of ion-specific electrodes, and Dr. Chou-Long Huang for his mentorship and support. This work was supported by the National Institutes of Health (K08DK091316 to ARR) and the American Society of Nephrology Gottschalk Award to ARR.

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The authors have nothing to disclose.

使用该拉姆齐检测，加上离子特定电极，允许液体分泌率和离子流的隔离昆虫马氏（肾）管的测量。二十个或更多小管可以一次进行测定，从而允许更高的吞吐量相比，个别的体外 microperfused小管的测定法。此外，离子特定电极允许在原位分泌液中的离子浓度的测定，从而限制了可以在小体积的流体至第二装置的转移而引入的误差。在协议中的关键步骤包括确保小管切开而不租金或撕裂（这将导致流体从撕裂分泌的，而不是输尿管的切断端），并确保在ISE工作良好通过确定事先其斜率在开始实验。即使采用小心解剖，它是不寻常的偶然肾小管失败分泌（高达肾小管〜10％解剖），但如果小管的高比例不分泌，这可能是对于生物学方面的原因（即，该特定的条件下，分泌率非常低）。一些缺陷，可能导致一个ISE都不尽如人意包括:离子载体漏出（其可能是由于电极末端断裂或硅烷化不足），或引入气泡进入尖端。这些问题可以通过在电极的可视化的化合物显微镜下确定，并通常要求在继续之前填充新的ISE与离子载体。此外，一个ISE可以很好地工作了好几天，甚至几周的时间，但最终会失败，再强制要求建设新的ISE。同样重要的是，化验盘是在实验结束时彻底冲洗，因为残余的盐将改变在随后的实验中的洗澡盐水的离子和渗透组合物。 根据我们的经验，Na +的ISE描述的这个协议是LESš稳定比第 K +的ISE，常常表现出它的斜率的劣化在实验的过程中。的确，Jayakannan 等人描述的改进的钠离子的ISE基于相同的离子载体具有更好的选择性和稳定性。他们的鸡尾酒包含3.5％4- 叔 -butylcalix [4]芳烃四乙酸四乙酯，95.9％2-硝基苯基辛基醚，和0.6％的钾，四（4-氯苯基）硼酸盐26。 一旦实验者是舒适进行这种测定，许多置换可以引入到探测上皮离子转运的根本生物学。例如，不同的基因型的小管可检查以确定的，或胞外或胞内信号传导途径调节这些转运或通道14,27跨上皮流体和离子迁移14的特定转运或离子通道的作用。洗澡液滴组合物可以被修改以看看其特定组件，诸如离子，葡萄糖或氨基酸9，或摩尔渗透压27,28的作用。或者，血淋巴本身从动物在不同条件下饲养得到可代替沐浴盐水16的使用。药物，激素（或它们的第二信使）或肽可以加入到确定其效果2,9,14,23,29。在目前的协议，在主段的流体和离子通量进行检查，由于初始段不分泌流体 2，和下段超出沐浴降。然而，该协议可以被修改来检查初始或低段30的活性。转基因记者，如钙敏感水母发光蛋白转基因5，或pH-和氯化物传感ChlopHensor转基因12，可以允许细胞内离子浓度与跨上皮通量的相关性。同样，Efetova 等。 cAMP水平的使用邻人证明小区固有操纵ptogenetic控制光敏腺苷酸环化酶，腺苷表明对星状和主单元31不同影响。 虽然目前的协议是专注于成年果蝇马氏管，所用的技术已经在其他昆虫得到应用，包括吸血蝽21，Hemideina毛利 24，以及多个蚊种17，以及对幼虫果蝇肾小管16。同样地，除了Na +和K +，如H +，钙离子或四乙基铵离子等可以测定30,32，以及毒素如水杨酸33。最后，离子特定电极技术也可以用于测量离子浓度microperfused哺乳动物小管，避免需要放射性标记的离子34,35。 总之，利用拉姆齐作为说用离子专用电极可以从孤立的果蝇跨上皮液和离子流的测量果蝇肾小管。再加上强大的果蝇遗传可用的工具，上皮细胞离子转运的分子机制可以阐明。

<html>肾上皮细胞离子转运underlies有机体iono-和渗透压调节。 果蝇马氏（肾）肾小管提供研究上皮细胞离子转运的分子机制提供了无与伦比的机会。这是由于果蝇的强大遗传学，配对的其肾小管生理研究的可访问的组合。拉姆齐检测，谁率先技术1研究者的名字命名，测量液体分泌率从孤立的马氏管，并建立在果蝇于1994年陶氏和他的同事2。这种利用果蝇的遗传工具，如GAL4-UAS系统3,4，确定细胞特异性的信号通路调节液分泌铺平了道路进一步研究。实例包括钙信号响应于肽激素5以及很多其他6,7。 ve_content"&gt;遗传技术和古典生理研究的组合表明，尿代在飞是通过从细管的主要链段的氯化钾富液的分泌。这是通过阳离子的平行跨上皮分泌，主要是K +也钠离子，通过主细胞，和Cl -通过星状细胞分泌8-12分别测量跨上皮K +和Na +通量的能力允许的传输机制比液体分泌的测量更详细的表征孤独。例如，在未刺激的果蝇细管为Na + / K + -ATP酶抑制剂哇巴因对分泌液2没有影响，即使当其摄取到主细胞是由有机阴离子转运抑制剂牛磺胆13抑制。然而，林顿和奥唐纳发现，哇巴因去极化基底外侧膜电位，并增加了钠离子通量9。如图中代表结果，我们重复这些发现，并表明，K +通量是伴随降低14;增大的 Na +通量和降低的 K +通量对液分泌相反的作用，造成在分泌无净的变化。因此，有两项决议的"乌本苷悖论"，即，哇巴因在果蝇小管的分泌液没有效果初步观察:首先，在刺激小管，哇巴因对液体分泌的效果并不明显，由于其吸收的有机阴离子转运13;第二，在未受刺激的小管，哇巴因一直反对对跨上皮Na +和K +流的影响，导致液分泌没有净变化（见代表性的成果和参考文献9）。因此， 钠离子的主要作用/ K + -ATP酶在未刺激的小管是降低细胞内Na +的浓度 ，以产生有利的浓度梯度为Na +的偶联的运输过程横跨基底外侧膜。事实上，通过分别测量Na +和K +通量，我们表明，小管缺乏飞钠-钾-2-氯转运蛋白（NKCC）具有降低的跨上皮的 K +通量，与哇巴因加入后没有进一步降低，并在经上皮无变化Na +的线 14。这些发现支持了我们的结论，即Na +的进入从而NKCC细胞通过的Na + / K + -ATP酶回收。在另一个例子中，Ianowski 等人观察到，降低浴的 K +浓度从10mM至6毫从吸血蝽下降跨上皮K +通量并增加经上皮Na +的磁通在小管，并在流体分泌没有净变化<sup&gt; 15。横跨幼虫小管上的 Na +通量和K +流的不同作用也已在果蝇小管中观察到响应于不同的盐饮食16并在两个蚊种响应于饲养盐度17。 在拉姆齐测定制备跨上皮离子通量的测定的最大挑战是分泌的流体中的离子浓度的测定。这个挑战已经取得了不同的解决方案，其中包括火焰photometery 18，使用放射性离子19和电子探针波谱仪20。这些技术需要分泌的液体滴转移到仪器的离子浓度的测量。因为流体的由未刺激的果蝇细管分泌的体积是小的，典型地〜0.5标升/分钟，这会带来一个技术挑战，也引入了错误，如果一些分泌的流体是在传输丢失。与此相反，使用离子特异性电极允许原位离子活性（从该离子浓度可以计算）的测量。目前的协议改编自所使用的Maddrell和同事使用缬氨霉素作为第 K +离子载体21测量通过吸血肾小管跨上皮的 K +通量，并且还描述了使用4- 叔 -butylcalix [4]芳烃-四乙酸的四乙酯基钠离子的特征在于Messerli 等特异性离子特异性电极。人。22。离子特异性电极也被用于测量离子浓度在流体中的拉姆齐测定在成人9,23和幼虫16 果蝇由马氏管分泌，新西兰阿尔派恩威塔（Hemideina毛利 ）24和在蚊子17。 在这里，我们详细描述了使用的拉姆齐作为说来测量流体分泌率在从果蝇马氏管，以及使用离子特异性电极以所分泌的流体中确定的 K + 和 Na +的浓度，从而跨上皮离子通量的计算。该试验的概述在图1中提供 。 <img alt="图1" src="/files/ftp_upload/53144/53144fig1.jpg" /> 图1.原理马氏管和拉姆齐分析与使用离子专用电极来测量离子浓度 。这个数字说明了该设置为拉姆齐检测。 （A）中的每个飞有四个小管，一对前管和一对后小管的，即浮在腹腔内通过血淋巴包围。在每一对中，两个小管联接在输尿管，然后在肠和hindgu交界清空尿吨。该管的盲端。尿被流体分泌主段（显示为红色）中产生，并朝向输尿管和流出到肠道内流动。解剖后，细管对被从肠道通过横断输尿管解离。 （B）的一对细管然后被转移到洗澡盐水的液滴内测定皿的孔中。其中的两个小管的，这里所指的"锚小管，"所缠绕的金属销和是惰性的。其他小管的分泌小管。起始段（其不分泌液）和分泌小管主体段保持洗澡盐水的液滴中。离子和水移动从洗澡盐水到主段的小管内腔，然后移向输尿管，正如在体内发生。下段（蓝色）是沐浴盐水，因此惰性外部。由于输尿管被切断时，分泌的流体出现从输尿管的切断端的液滴。 Ť他分泌流体液滴放大随时间而分泌的继续，其直径是使用目镜测微尺测定。矿物油的层防止分泌流体的蒸发。参考和离子专用电极测量分泌液的离子浓度。 <a href="https://www.jove.com/files/ftp_upload/53144/53144fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table><tbody><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Ellsworth Adhesives</td><td>http://www.ellsworth.com/dow-corning-sylgard-184-silicone-encapsulant-0-5kg-kit-clear/</td><td>May be purchased from multiple distributors</td></tr><tr><td>Petri dish, polystyrene, 100 mm x 15 mm</td><td>Fisher</td><td>FB0875712</td><td>Specific brand is not important</td></tr><tr><td>Petri dish, polystyrene, 35 mm x 10 mm</td><td>Corning Life Sciences</td><td>Fisher 08-757-100A</td><td>Specific brand is not important</td></tr><tr><td>Scalpel Handle #3</td><td>Fine Science Tools</td><td>10003-12</td><td>Specific brand is not important</td></tr><tr><td>Scalpel Blades #1</td><td>Fine Science Tools</td><td>10011-00</td><td>Specific brand is not important; use appropriate sharps precautions</td></tr><tr><td>Needle, 30 G x 1/2</td><td>Becton Dickinson</td><td>305106</td><td>Use appropriate sharps precautions</td></tr><tr><td>Minutien pins, black anodized, 0.15 mm</td><td>Fine Science Tools</td><td>26002-15</td><td></td></tr><tr><td>Stereomicroscope with ocular micrometer</td><td>Nikon</td><td>SMZ800</td><td>Specific brand is not important; this is given as an example</td></tr><tr><td>Sheet of black stained glass, 3 mm (1/8 inch) thick</td><td>Hobby shop</td><td></td><td>Example includes Spectrum Black Opal by Spectrum Glass (http://www.delphiglass.com/spectrum-glass/opalescent/spectrum-black-opal)</td></tr><tr><td>Glass cutting tools (glass cutter, glass cutting pliers)</td><td>Hobby shop</td><td></td><td>Examples include the Studio Pro Lightweight Running Pliers by Diamond Tech (http://www.delphiglass.com/glass-cutters-tools/pliers-nippers/studio-pro-lightweight-running-pliers) and the Studio Pro Brass Glass Cutter by Diamond Tech (http://www.delphiglass.com/glass-cutters-tools/glass-cutters/studio-pro-brass-glass-cutter). Use appropriate safety precautions when cutting glass</td></tr><tr><td>Borosilicate glass capillary tube, unfilamented, GC120-10, OD 1.2 mm, ID 0.69 mm, length 10 cm</td><td>Warner Instruments</td><td>30-0042</td><td></td></tr><tr><td>Borosilicate glass capillary tube, filamented, GC120F-10, OD 1.2 mm, ID 0.69 mm, length 10 cm</td><td>Warner Instruments</td><td>30-0044</td><td></td></tr><tr><td>Nitric acid, 70%</td><td>Sigma</td><td>438073</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines. Specific brand is not important</td></tr><tr><td>Cimarec 7 in x 7 in hotplate</td><td>Fisher</td><td>11675911Q</td><td>Specific brand is not important; caution when heated</td></tr><tr><td>Selectophore dichlorodimethylsilane</td><td>Sigma</td><td>40136-1ML</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Two-step vertical pipet puller</td><td>Narishige</td><td>PC-10</td><td>Other pipet pullers can be used; this is given as an example</td></tr><tr><td>Glass petri dish, 150 mm diameter x 15 mm height</td><td>Fisher</td><td>08-748E</td><td>Specific brand is not important; only one dish needed</td></tr><tr><td>World Precision Instruments E210 1 mm micropipette storage jar</td><td>Fisher</td><td>50-821-852</td><td>May be available from other distributors. Useful to have two jars. Note that although this jar is specified for 1 mm pipets, and the pipets used here are 1.2 mm, in our experience the 1 mm jar works best for the 1.2 mm pipets.</td></tr><tr><td>Silica Gel, Tel-Tale Desiccant, indicating, 10-18 mesh</td><td>Fisher</td><td>S161-500</td><td>Indicating silica useful for determining whether silica gel retains desiccating ability</td></tr><tr><td>World Precision Instruments MicroFil, 34G</td><td>Fisher</td><td>50-821-914</td><td>May be available from other distributors.</td></tr><tr><td>1 ml syringe with luer lock</td><td>Becton Dickinson</td><td>309659</td><td>May be available from other distributors.</td></tr><tr><td>3 ml syringe with luer lock</td><td>Becton Dickinson</td><td>309657</td><td>May be available from other distributors.</td></tr><tr><td>D300 3-way stopcock with female luer lock inlet port, male luer outlet port with rotating collar and guard</td><td>Cole-Parmer</td><td>UX-30600-02</td><td>Specific brand is not important</td></tr><tr><td>Female Luer Locking Connector</td><td>4 Medical Solutions</td><td>ADC 9873-10</td><td>Specific brand is not important; barbed end is ~4 mm at narrowest point and ~7 mm at widest point.</td></tr><tr><td>Silicone Tubing I.D. x O.D. x Wall: 1/16 x 1/8 x 1/32 in. (1.59 x 3.18 x 0.79 mm)</td><td>Fisher</td><td>14-179-110</td><td>Specific brand is not important</td></tr><tr><td>E-3603 tubing, I.D. x O.D.: 1/32 x 3/32 in</td><td>Fisher</td><td>14171208</td><td>Specific brand is not important</td></tr><tr><td>Modeling clay</td><td></td><td></td><td>Specific brand is not important</td></tr><tr><td>Selectophore potassium ionophore I, cocktail B</td><td>Sigma</td><td>99373</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Selectophore sodium ionophore X</td><td>Sigma</td><td>71747</td><td>Sodium ionophore X = 4-tert-butylcalix[4]arene-tetraacetic acid tetraethylester</td></tr><tr><td>Selectophore 2-nitrophenyl octyl ether</td><td>Sigma</td><td>73732</td><td></td></tr><tr><td>Selectophore sodium tetraphenylborate</td><td>Sigma</td><td>72018</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Schneider&#039;s Drosophila medium</td><td>Life Technologies</td><td>21720024</td><td></td></tr><tr><td>High impedance electrometer</td><td>World Precision Instruments</td><td>FD223a</td><td></td></tr><tr><td>Microelectrode holder 1 mm with 45&amp;deg; body, vented, with handle</td><td>Warner Instruments</td><td>64-1051</td><td></td></tr><tr><td>Microelectrode holder 1 mm with straight body, vented</td><td>Warner Instruments</td><td>64-1007</td><td></td></tr><tr><td>Silver wire</td><td>Warner Instruments</td><td>64-1318</td><td></td></tr><tr><td>Micromanipulators, pair</td><td>Leitz</td><td></td><td>Various brands/models will work; this is an example</td></tr><tr><td>Faraday cage</td><td>Technical Manufacturing Corporation</td><td>81-334-03</td><td>This is an example; any Faraday cage will work</td></tr><tr><td>Single gooseneck fiberoptic light</td><td>Nikon</td><td></td><td>Specific brand is not important</td></tr><tr><td>mineral oil</td><td>Fisher</td><td>BP-2629</td><td>Specific brand is not important</td></tr><tr><td>forceps, Dumont #5 with Biologie tip</td><td>Fine Science Tool</td><td>11295-10</td><td>May be available from other distributors.</td></tr></tbody></table>

use of the ramsay assay to measure fluid secretion and ion flux rates in the drosophila melanogaster malpighian tubule

肾小管上皮离子转运的调制允许生物体维持离子和渗透压稳态变化的外部条件的面部。 果蝇马氏（肾）肾小管提供研究上皮细胞离子转运的分子机制，因为这种微生物及其肾小管生理研究的辅助强大的遗传学一个无与伦比的机会。在这里，我们描述了使用的拉姆齐测定来测量流体分泌率从分离的飞肾小管，与使用离子特异性电极测量钠和钾的浓度，在分泌的流体。该测定允许研究的跨上皮流体和离子通量〜20小管的时间，而不需要在分泌的流体传送到一个​​单独的装置来测量离子浓度。遗传上不同的小管可以分析，以评估在运输过程特定基因的作用。另外，将bathing盐水可以被修改来检查添加其化学特性，或药物或激素的作用。综上所述，本技术允许的上皮离子输送这些传输机制中的果蝇细管，以及调控基本机制的分子表征。

图7和8表明，使用拉姆齐测定用离子特异性电极来测量的 K + 和 Na +浓度可以区分基因和药理学上不同的 K + 和 Na +通量，即不通过单独测量流体分泌率捕获的信息。 图图7示出了从果蝇携带在NKCC纯合无效突变降低流体分泌小管是通过以K +流中的突变体（图7A）的下降驱动，而Na +的磁通没有改变（图7B）。 图8示出了该钠 / K + -ATP酶抑制剂，乌本苷，对每个测量的三个参数的不同的影响:在野生型小管，流体分泌速率不变（图8A和8B），Na +的磁通增加（图8A ）和K +流是德折痕（图8B）。此外，哇巴因具有在NKCC 无效突变小管（图8B）上的跨上皮K +磁通没有影响。总之，这些数据表明， 钠 / K + -ATP酶提供驱动力为K +通过NKCC摄取，而Na +的通过的 Na + / K + -ATP酶14再循环。 13至18小管是在各基因型/条件分析;观察到的差异是与AP &lt;0.01（或更少）统计学显著通过未配对，双尾Student t检验。因为〜15-20小管可以很容易地在一天的实验进行分析，这里显示的结果代表〜10日的实验。 <img alt="图7" src="/files/ftp_upload/53144/53144fig7.jpg" /> 图7.液分泌速率和跨上皮ķ <st荣&gt; + 通量下降，肾小管携带纯合子无效突变的NKCC，而钠离子通量不变。（A）液分泌率（NL /分钟/小管）和K +流 （皮摩尔/分钟/小管）进行测定从控制苍蝇（wBerlin）或携带苍蝇在飞NKCC纯合无效突变小管（W; Ncc69 R2 / Ncc69 R2）。 N = 18管/基因型。 （B）液分泌率（NL /分钟/小管）和Na +流 （皮摩尔/分钟/小管）从控制果蝇（wBerlin，N = 13管），或从苍蝇携带纯合子无效突变在飞NKCC（测瓦; Ncc69 R2 / Ncc69 R2，N = 14管）。所示值为平均值（SEM）的平均值±标准误差。 NS，不显著; **，P &lt;0.01;***，P &lt;0.001; ****，P &lt;0.0001。这个数字是改编自罗丹等。人 14 <a href="https://www.jove.com/files/ftp_upload/53144/53144fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/53144/53144fig8.jpg" /> 图8.的Na + / K + -ATP酶回收的 Na +进入通过NKCC。（A）中的流体分泌速率（升/分钟/小管） 和 Na +通量（皮摩尔/分钟/小管），测定在从控制蝇肾小管（wBerlin）中的100μM的哇巴因，所述钠 / K + -ATP酶的抑制剂的情况下（N = 13小管）或存在（15小管）。 （B）的流体分泌速率（升/分钟/小管） 和 K + </sup&gt;通量（皮摩尔/分钟/小管）的测量管从控制苍蝇（wBerlin）在100微米哇巴因缺乏（N = 17管）或存在（N = 17管）解剖。 K +通量还测量在在没有或哇巴因的存在NKCC 空管（瓦特; Ncc69 R2 / Ncc69 R2中，n = 16 - 17细管/条件）。显示值是平均值±标准差。 NS，不显著; **，P &lt;0.01; ***，P &lt;0.001。这个数字是改编自罗丹等。人 14 <a href="https://www.jove.com/files/ftp_upload/53144/53144fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table><tbody><tr><td colspan="2">果蝇生理盐水</td><td></td><td></td></tr><tr><td>对于1L: </td><td></td><td></td><td></td></tr><tr><td colspan="4">称出3.6克的葡萄糖，并放入一个烧杯中;最终的合作ncentration = 20毫</td></tr><tr><td>然后添加: </td><td></td><td></td><td></td></tr><tr><td>零件</td><td>体积（ml） </td><td colspan="2">终浓度（MM） </td></tr><tr><td> H 2 O的</td><td> 〜800 </td><td></td><td></td></tr><tr><td> 2.5 M氯化钠</td><td> 47 </td><td> 117.5 </td><td></td></tr><tr><td> 1米氯化钾</td><td> 20 </td><td> 20 </td><td></td></tr><tr><td> 1M的氯化钙 </td><td> 2 </td><td> 2 </td><td></td></tr><tr><td> 1M的氯化镁 </td><td> 8.5 </td><td> 8.5 </td><td></td></tr><tr><td> 1M的碳酸氢钠 </td><td> 10.2 </td><td> 10.2 </td><td></td></tr><tr><td>溶于1M HEPES </td><td> 15 </td><td> 15 </td><td></td></tr><tr><td>的1M的NaH 2 PO 4 </td><td> 4.3 </td><td> 4.3 </td><td></td></tr><tr><td colspan="2">搅拌均匀，调节pH值至7.0 </td><td></td><td></td></tr><tr><td colspan="2">加入H 2 O操作1L的最终体积</td><td></td><td></td></tr><tr><td colspan="4">过滤消毒;避免高压灭菌（葡萄糖会焦糖） </td></tr><tr><td>储存在4℃ </td><td></td><td></td><td></td></tr><tr><td colspan="3">如果丢弃的证据细菌或真菌生长</td><td></td></tr></tbody></table> 表1.使果蝇盐水。 <table><tbody><tr><td>施耐德的媒体</td><td></td></tr><tr><td>零件</td><td>浓度（mM） </td></tr><tr><td>甘氨酸</td><td> 3.33 </td></tr><tr><td> L-精氨酸</td><td> 2.3 </td></tr><tr><td> L-天冬氨酸</td><td> 3.01 </td></tr><tr><td> L-半胱氨酸</td><td> 0.496 </td></tr><tr><td> L-胱氨酸</td><td> 0.417 </td></tr><tr><td> L-谷氨酸</td><td> 5.44 </td></tr><tr><td> L-谷氨酰胺</td><td> 12.33 </td></tr><tr><td> L-组氨酸</td><td> 2.58 </td></tr><tr><td> L-异亮氨酸</td><td> 1.15 </td></tr><tr><td> L-亮氨酸</td><td> 1.15 </td></tr><tr><td> L-赖氨酸盐酸盐</td><td> 9.02 </td></tr><tr><td> L-蛋氨酸</td><td> 5.37 </td></tr><tr><td> L-苯丙氨酸</td><td> 0.909 </td></tr><tr><td> L-脯氨酸</td><td> 14.78 </td></tr><tr><td> L-丝氨酸</td><td> 2.38 </td></tr><tr><td> L-苏氨酸</td><td> 2.94 </td></tr><tr><td> L-色氨酸</td><td> 0.49 </td></tr><tr><td> L-酪氨酸</td><td> 2.76 </td></tr><tr><td> L-缬氨酸</td><td> 2.56 </td></tr><tr><td> β丙氨酸</td><td> 5.62 </td></tr><tr><td> 氯化钙 </td><td> 5.41 </td></tr><tr><td> 硫酸镁 </td><td> 15.06 </td></tr><tr><td>氯化钾</td><td> 21.33 &lt;/ TD&gt; </tr><tr><td> 磷酸二氢钾 </td><td> 3.31 </td></tr><tr><td>氯化钠</td><td> 36.21 </td></tr><tr><td> 碳酸氢钠 </td><td> 4.76 </td></tr><tr><td> 的 Na 2 HPO 4 </td><td> 4.94 </td></tr><tr><td> α-酮戊二酸</td><td> 1.37 </td></tr><tr><td> D-葡萄糖</td><td> 11.11 </td></tr><tr><td>富马酸</td><td> 0.862 </td></tr><tr><td>苹果酸</td><td> 0.746 </td></tr><tr><td>琥珀酸</td><td> 0.847 </td></tr><tr><td>海藻糖</td><td> 5.85 </td></tr><tr><td>酵母自溶液</td><td> 2000毫克/升</td></tr></tbody></table> 表2.组成施耐德的培养基。 <table><tbody><tr><td colspan="2">预</td><td colspan="2">帖子</td><td> 间隔 </td><td> 斜率/等分 </td></tr><tr><td> 毫</td><td> 毫伏 </td><td> 毫 </td><td> 毫伏 </td><td> 15 - 150（预） </td><td> 52.8 </td></tr><tr><td> 15 </td><td> -28.4 </td><td> 15 </td><td> -29.4 </td><td> 15 - 150（后） </td><td> 54.4 </td></tr><tr><td> 75 </td><td> 9.1 </td><td> 75 </td><td> 9.4 </td><td> 75 - 150（预） </td><td> 51 </td></tr><tr><td> 150 </td><td> 24.4 </td><td> 150 </td><td> 25 </td><td> 75 - 150（后） </td><td> 52 </td></tr><tr><td> 200 </td><td> 30.6 </td><td> 200 </td><td> 31.9 </td><td> 150 - 200（预） </td><td> 49.6 </td></tr><tr><td></td><td></td><td></td><td></td><td> 150 - 200（后） </td><td> 55.2 </td></tr><tr><td></td><td></td><td></td><td></td><td> 意味着 </td><td> 52.5 </td></tr></tbody></table> 表3.校准的K </stron克&gt; + ISE K +的ISE校准在含有15，75，150和200毫米氯化钾之前的解决方案和实验分泌流体的测量之后。实际测量的一个例子示于该表中。在K +浓度的斜率/等分变化的计算说明。对于15-150 mV的区间，斜率/等分= MV，对于150毫米校准下降 - 毫伏15毫米校准下降。为75 - 150间隔，所不同的是由0.3 [日志（七十五分之一百五十零）]除以。为150 - 200间隔，所不同的是由0.125 [日志（200/150）]除以。六坡（三级预实验，三后实验）进行平均，以获得平均坡度/等分。该表改编自罗丹等。人 14

Watch this Scientific Journal Video about Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule at JoVE.com

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

这个协议描述了使用的拉姆齐测定来测量流体分泌率从果蝇孤立马氏（肾）细管。此外，使用离子特异性电极来测量分泌的流体钠和钾的浓度，允许跨上皮离子通量的计算，进行说明。

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 ...

use-ramsay-assay-to-measure-fluid-secretion-ion-flux-rates-drosophila

Nanyang Technological University

Research

JoVE Journal

Biology

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

11.8K 次观看.  University of Texas Southwestern Medical Center. 这个协议描述了使用的拉姆齐测定来测量流体分泌率从果蝇孤立马氏（肾）细管。此外，使用离子特异性电极来测量分泌的流体钠和钾的浓度，允许跨上皮离子通量的计算，进行说明。 

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

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

对检测方法果蝇行为

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

科研

神经科学

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

测量的生理反应<em&gt;果蝇</em&gt;感觉神经元脂质费洛蒙使用实时钙成像

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

免疫与感染

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

毛细管馈线分析测量的摄食<em&gt;果蝇</em

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

幼虫活动的测量<em&gt;果蝇</em&gt;活动监视器

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

生物学

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

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

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

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

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 ...

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.

This protocol outlines methodologies behind the Ramsay assay, ion-selective microelectrodes, Scanning Ion-selective Electrode Technique (SIET) and in vitro contraction assays, applied to study the adult mosquito excretory system, comprised of the Malpighian tubules and hindgut, to collectively measure ion and fluid secretion rates, contractile activity, and transepithelial ion transport.

研究成年蚊子排泄系统神经肽和其他调节剂的活性

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the ...

Monitoring Gut Acidification in the Adult Drosophila Intestine

The fruit fly midgut consists of multiple regions, each of which is composed of cells that carry out unique physiological functions required for the proper functioning of the gut. One such region, the copper cell region (CCR), is localized to the middle midgut and consists, in part, of a group of cells known as copper cells. Copper cells are involved in gastric acid secretion, an evolutionarily conserved process whose precise role is poorly understood. This paper describes improvements in the current protocol used to assay for acidification of the adult Drosophila melanogaster gut and demonstrates that it can be used on other species of flies. In particular, this paper demonstrates that gut acidification is dependent on the fly's nutritional status and presents a protocol based on this new finding. Overall, this protocol demonstrates the potential usefulness of studying Drosophila copper cells to uncover general principles underlying the mechanisms of gut acidification.

Monitoring Gut Acidification in the Adult Drosophila Intestine

Here, we present a standardized protocol for monitoring gut acidification in Drosophila melanogaster with optimal output. We first use this protocol for gut acidification monitoring in Drosophila melanogaster and then demonstrate its use in non-model Drosophila species.

监测成人 果蝇 肠道的肠道酸化

The fruit fly midgut consists of multiple regions, each of which is composed of cells that carry out unique physiological functions required for the ...

果蝇 用于药物发现的粪便沉积物分析

Assessment of The Effect of Antidiarrheal Drugs and Plant Extracts on Drosophila melanogaster

To study human gastrointestinal physiology, biomedical scientists have relied on the use of model organisms. Although many researchers have used mice as a model to study intestinal function, only a few reports have focused on Drosophila melanogaster (D. melanogaster). Compared to mice, fruit flies present many advantages, such as a short life cycle, cost-effective and simple maintenance, and no ethical issues. Furthermore, the mammalian gastrointestinal physiology, anatomy, and signaling pathways are highly conserved in D. melanogaster. Plant extracts have been used traditionally to treat diarrhea and constipation. For example, Psidium guajava (P. guajava) is one of the most known antidiarrheal agents in the tropics. However, no studies have evaluated the effect of antidiarrheal and laxative drugs and plant extracts in D. melanogaster, and it remains unknown if similar effects (e.g., smaller, more concentrated, and less abundant fecal deposits in the case of antidiarrheal drugs) can occur in the fruit flies compared to mammals. In this study, an antidiarrheal effect induced by P. guajava is demonstrated in a D. melanogaster strain that presents a diarrheic phenotype. Fecal sampling produced by flies is monitored using a dye-supplemented food. This protocol outlines the method used for preparing food with drugs, evaluating the fecal deposits of flies fed on these food preparations, and interpreting the data obtained.

作者聚焦：利用果蝇模型推进止泻药研究 

Here, a method is described to feed Drosophila melanogaster with drugs and plant extracts and assess their effect on the gastrointestinal tract by analyzing the fruit flies fecal deposits. The drug-treated flies can be used as a model for further research.

止泻药和植物提取物对黑腹果蝇效果的评估

To study human gastrointestinal physiology, biomedical scientists have relied on the use of model organisms. Although many researchers have used mice as a ...

使用拉姆齐试验来测量液体分泌和离子流率在

摘要

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此视频中的章节

对检测方法果蝇行为

测量的生理反应

毛细管馈线分析测量的摄食

幼虫活动的测量

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

研究成年蚊子排泄系统神经肽和其他调节剂的活性

监测成人果蝇肠道的肠道酸化

止泻药和植物提取物对黑腹果蝇效果的评估

止泻药和植物提取物对黑腹果蝇效果的评估

止泻药和植物提取物对黑腹果蝇效果的评估

使用拉姆齐试验来测量液体分泌和离子流率在

摘要

探索更多视频

此视频中的章节

对检测方法果蝇行为

测量的生理反应

毛细管馈线分析测量的摄食

幼虫活动的测量

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

研究成年蚊子排泄系统神经肽和其他调节剂的活性

监测成人 果蝇 肠道的肠道酸化

止泻药和植物提取物对黑腹果蝇效果的评估

止泻药和植物提取物对黑腹果蝇效果的评估

止泻药和植物提取物对黑腹果蝇效果的评估

监测成人果蝇肠道的肠道酸化