Name: monitoring gut acidification in the adult drosophila intestine
Uploaded: 2021-10-11T14:00:00
Description: Watch this Scientific Journal Video about Monitoring Gut Acidification in the Adult Drosophila Intestine at JoVE.com

作者承认，对作者实验室工作的支持由HHMI教师学者奖和UT西南医学中心儿童研究所的启动资金提供。

注：标准实验室线俄勒冈R被用作WT对照。所有苍蝇在标准玉米粉 - 糖蜜培养基（含有糖蜜，琼脂，酵母，玉米粉，癡脂，丙酸和水）下在室温下以12/12小时的浅/暗昼夜节律饲养。
1. 检测准备
<ol><li>在CO2 麻醉下收集雌性苍蝇（0-2天大，非处女），并让它们在实验前至少在标准玉米粉食物中恢复3天。</li>
	<li>在室温（〜23°C）下将苍蝇饿死在含有用〜2mL去?...

<ol>
	<li>Hollander, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+composition+and+mechanism+of+formation+of+gastric+acid+secretion.">The composition and mechanism of formation of gastric acid secretion.</a> Science. 110 (2846), 57-63 (1949).</li><li>Forte, J. G., Zhu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apical+recycling+of+the+gastric+parietal+cell+H,+K-ATPase.">Apical recycling of the gastric parietal cell H, K-ATPase.</a> Annual Review of Physiology. 72, 273-296 (2010).</li><li>Samuelson, L. C., Hinkle, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+regulation+of+gastric+acid+secretion+through+analysis+of+genetically+engineered+mice.">Insights into the regulation of gastric acid secretion through analysis of genetically engineered mice.</a> Annual Review of Physiology. 65, 383-400 (2003).</li><li>Yao, X., Forte, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology+of+acid+secretion+by+the+parietal+cell.">Cell biology of acid secretion by the parietal cell.</a> Annual Review of Physiology. 65, 103-131 (2003).</li><li>Driver, I., Ohlstein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+regional+intestinal+stem+cell+identity+during+Drosophila+metamorphosis.">Specification of regional intestinal stem cell identity during Drosophila metamorphosis.</a> Development. 141 (9), 1848-1856 (2014).</li><li>Overend, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+and+functional+significance+of+acid+generation+in+the+Drosophila+midgut.">Molecular mechanism and functional significance of acid generation in the Drosophila midgut.</a> Scientific Reports. 6, 27242 (2016).</li><li>Shanbhag, S., Tripathi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+ultrastructure+and+cellular+mechanisms+of+acid+and+base+transport+in+the+Drosophila+midgut.">Epithelial ultrastructure and cellular mechanisms of acid and base transport in the Drosophila midgut.</a> Journal of Experimental Biology. 212, 1731-1744 (2009).</li><li>Dubreuil, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper+cells+and+stomach+acid+secretion+in+the+Drosophila+midgut.">Copper cells and stomach acid secretion in the Drosophila midgut.</a> International Journal of Biochemistry and Cell Biology. 36 (5), 745-752 (2004).</li><li>Martorell, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+mechanisms+of+tumorigenesis+in+the+Drosophila+adult+midgut.">Conserved mechanisms of tumorigenesis in the Drosophila adult midgut.</a> PLoS ONE. 9 (2), 88413 (2014).</li><li>Strand, M., Micchelli, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+control+of+Drosophila+gut+stem+cell+proliferation:+EGF+establishes+GSSC+proliferative+set+point+&amp;+controls+emergence+from+quiescence.">Regional control of Drosophila gut stem cell proliferation: EGF establishes GSSC proliferative set point &amp; controls emergence from quiescence.</a> PLoS One. 8 (11), 80608 (2013).</li><li>Storelli, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+perpetuates+nutritional+mutualism+by+promoting+the+fitness+of+its+intestinal+symbiont+Lactobacillus+plantarum.">Drosophila perpetuates nutritional mutualism by promoting the fitness of its intestinal symbiont Lactobacillus plantarum.</a> Cell Metabolism. 27 (2), 362-377 (2018).</li><li>Abu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Communicating+the+nutritional+value+of+sugar+in+Drosophila.">Communicating the nutritional value of sugar in Drosophila.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (12), 2829-2838 (2018).</li><li>Blecker, U., Gold, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastritis+and+ulcer+disease+in+childhood.">Gastritis and ulcer disease in childhood.</a> European Journal of Pediatrics. 158 (7), 541-546 (1999).</li></ol>

该协议中的一个关键步骤是正确解剖肠道，以可视化酸化表型的CCR。当肠道完好无损时，从铜细胞释放的酸被限制在CCR中。然而，在解剖过程中，由肠道破裂引起的泄漏可导致酸从CCR扩散，并导致肠道被错误地评为酸化阴性。此外，指示酸化的黄色在解剖后5-10分钟内褪色，强调了分离后不久对肠道酸化表型进行评分的重要性。最后，目前的方案7，8...

在昆虫肠道中，铜细胞与哺乳动物胃的产酸胃壁细胞（也称为oxyntic）具有细胞和功能相似性。这组细胞将酸释放到肠腔中。酸分泌和解剖学的功能在进化上是保守的。排放的酸的主要成分是盐酸和氯化钾。细胞中酸形成的化学机制取决于碳酸酐酶。这种酶从CO2 和水中产生碳酸氢根离子，释放出一个羟基离子，然后通过质子泵排入腔内以换取钾。氯离子和钾离子通过电导通道输送到管腔中，形成盐酸和氯化钾，氯化钾是胃液的主要成分1，2，3，4。
虽然酸形成的机制已经很清楚了，但对调节酸分泌的生理机制知之甚少。开发这种方法的目的是帮助更好地描绘协调酸形成和分泌的细胞途径，并确定酸在介导肠道生理学和稳态中的作用。开发和使用该技术背后的基本原理是提供一种一致且可靠的方法来研究 果蝇 和非模型生物的肠道酸化过程。虽然目前存在用于测定 果蝇 中谷酸化的标准方案2，5，6，但在使用该方案研究铜细胞功能时，观察到野生型（WT）苍蝇酸化程度的显着变异性。为了理解这种观察到的变异性的基础并获得一致的结果，如下所述优化了标准协议的几个方面。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>B0126</td>
		</tr>
		<tr>
			<td>cellSens software</td>
			<td>Olympus</td>
			<td></td>
			<td>Image aqusition (https://www.olympus-lifescience.com/en/software/cellsens)</td>
		</tr>
		<tr>
			<td>D. simulans</td>
			<td>Drosophila Species Stock Center at the University of California</td>
			<td></td>
			<td>Riverside California1 (https://www.drosophilaspecies.com/)</td>
		</tr>
		<tr>
			<td>D. erecta</td>
			<td>Drosophila Species Stock Center at the University of California</td>
			<td></td>
			<td>Dere cy1(https://www.drosophilaspecies.com/)</td>
		</tr>
		<tr>
			<td>D. pseudoobscura</td>
			<td>Drosophila Species Stock Center at the University of California</td>
			<td></td>
			<td>Eugene, Oregon(https://www.drosophilaspecies.com/)</td>
		</tr>
		<tr>
			<td>D. mojavensis</td>
			<td>Drosophila Species Stock Center at the University of California</td>
			<td></td>
			<td>Chocolate Mountains, California (https://www.drosophilaspecies.com/)</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Inox Biology</td>
			<td></td>
			<td>Catalog# 11252-20</td>
		</tr>
		<tr>
			<td>Fuji</td>
			<td>Fuji</td>
			<td></td>
			<td>Image processing (https://hpc.nih.gov/apps/Fiji.html)</td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>VWR</td>
			<td></td>
			<td>Catalog#16005-108</td>
		</tr>
		<tr>
			<td>Kim wipes Tissue</td>
			<td>Kimtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope and camera</td>
			<td>Olympus SZ61 microscope equipped with an Olympus D-27 digital camera</td>
			<td></td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>Oregon R</td>
			<td>Bloomington Drosophila Stock</td>
			<td></td>
			<td>(https://bdsc.indiana.edu/ # 2376)</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>Catalog #FB0875713A</td>
		</tr>
		<tr>
			<td>Phosphate-buffered Saline (PBS)</td>
			<td>HyClone</td>
			<td></td>
			<td>Catalog # SH30258.01</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Olympus SZ51</td>
			<td></td>
			<td>Visual magnification</td>
		</tr>
	</tbody>
</table>

monitoring gut acidification in the adult drosophila intestine

果蝇中肠由多个区域组成，每个区域都由执行肠道正常功能所需的独特生理功能的细胞组成。一个这样的区域，铜细胞区域（CCR），定位于中肠中部，部分由一组称为铜电池的细胞组成。铜细胞参与胃酸分泌，这是一种进化保守的过程，其确切的作用知之甚少。本文描述了目前用于测定成年 果蝇 肠道酸化的方案的改进，并证明它可以用于其他种类的苍蝇。特别是，本文表明肠道酸化取决于苍蝇的营养状况，并提出了基于这一新发现的方案。总体而言，该协议证明了研究 果蝇 铜细胞的潜在有用性，以揭示肠道酸化机制背后的一般原理。

我们使俄勒冈R雌性苍蝇挨饿超过20小时，然后喂它们补充了BPB（2%）的食物约12小时，如前所述7，8，9，10，11。溴酚蓝（BPB）是一种pH传感染料。它在pH 3.0时从黄色变为pH 4.6及以上时变为蓝色。如前所述，在肠道解剖后，发现一些苍蝇产生酸，如肠道CCR中的黄色所示（<strong cl...

Watch this Scientific Journal Video about Monitoring Gut Acidification in the Adult Drosophila Intestine at JoVE.com

Monitoring Gut Acidification in the Adult Drosophila Intestine

在这里，我们提出了一种标准化的方案，用于监测 黑腹果蝇 的肠道酸化，并具有最佳输出。我们首先将此方案用于 黑色果蝇 的肠道酸化监测，然后证明其在非模型 果蝇 物种中的使用。

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 ...

这是监测果蝇肠道酸化的最快和最正确的方案，具有强大而真实的结果。这种方法将有助于确定肠道酸化在多种生物体中的作用。而这种现象就是这种技术的简单性是主要优点。因此，它可以用于非模式生物。该方法可以监测许多系统的酸释放，包括细胞系和活培养物上的3D。这种技术很简单，而且用最少的力气。含有溴酚蓝的食物应新鲜烹制。此外，虽然解剖肠道不应拉伸。开始安排肠道酸化监测测定，用pH传感溴酚蓝或BPB染料制备苍蝇食物。为此，将苍蝇食物放入微波炉中融化，让它冷却直至温热。将一毫升4%BPB加入一毫升温热食品中，搅拌均匀。使用移液器，将含有BPB的苍蝇食物添加到培养皿中心的约200微升的单点中。接下来，收集0至2天大的非处女雌性果蝇黑色素蝇，并让苍蝇在标准玉米粉食物中恢复。在实验之前，在室温下将苍蝇饿死在装有约两毫升去离子水的实验室擦拭组织的小瓶中24小时。将饥饿的苍蝇转移到含有新鲜制备的苍蝇食物的单点的培养皿中，补充2%BPB，让苍蝇在室温下觅食四小时，同时暴露在光线下。四小时后收集苍蝇，然后在体视显微镜下用镊子在1X PBS中手术分离麻醉苍蝇的内脏。计算显示强大的BPB站在肠道中的苍蝇的数量，并使用等式计算百分比。解剖后，将PBS中的样品安装到载玻片上，并将准备好的载玻片放在显微镜下。然后使用 IPS 调整焦点。完成后，关闭IPS以打开相机的快门。在肠道解剖后，发现一些果蝇黑色素食蝇雌性苍蝇产生酸，如肠道CCR中的黄色所示。而一些苍蝇的肠子是蓝色的，这表明苍蝇未能酸化它们的肠道。在30分钟内，大约20%的苍蝇使肠道酸化。一小时后，40%的肠道内脏显示酸化，而在喂食两到三个小时后，酸化内脏的百分比增加到60%至70%当苍蝇喂食四小时时，几乎90%至95%的内脏被酸化。在两种不同的温度条件下，没有观察到肠道酸化的差异。在各种果蝇物种中评估了肠道酸化方案的效率。所有测试物种都显示出强烈的肠道酸化，这表明该协议可以应用于其他生物体。要记住的最重要的事情是将星蝇放入带有酵母和溴酚蓝的培养皿中四个小时。该技术将有助于了解参与肠道酸化的基本生物学和细胞途径。它还应该有助于确定药物靶点，以产生与肠道酸化相关的疾病。

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

Monitoring Gut Acidification in the Adult Drosophila Intestine

Unit 1

Membrane Transport and Active Transporters

SCIENTISTS IN ACTION

Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes

Extracting DNA from the Gut Microbes of the Termite Zootermopsis Angusticollis and Visualizing Gut Microbes

从白蚁肠道微生物（Zootermopsis Angusticollis）中提取的DNA和可视化肠道微生物

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and ...

科研

生物学

Whole Cell Recordings from Brain of Adult Drosophila

从整个成年果蝇大脑细胞记录

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

高分辨率视频跟踪移动的，在成年果蝇。

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

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  ...

Dissection of Oenocytes from Adult Drosophila melanogaster

Dissection of Oenocytes from Adult Drosophila melanogaster

从成人Oenocytes剖析果蝇</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

注射胰岛素和血淋巴的提取来衡量成人胰岛素敏感性果蝇</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像<em&gt;果蝇</em&gt;感光细胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Gavaging Adult Zebrafish

Gavaging成年斑马鱼

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

监测成人 果蝇 肠道的肠道酸化

监测成人果蝇肠道的肠道酸化