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.

함께 특정한 이온 전극 램지 분석의 사용은, 절연 곤충 Malpighian (신장)에서 유체 세뇨관 분비 속도 및 이온 플럭스를 측정 할 수있다. 스물 이상의 세관은 체외 microperfused 세뇨관에서 개별의 분석법에 비해 더 높은 처리량을 가능하게 한 번에 분석 될 수있다. 또한, 이온 전극을 특정 제 2 장치로의 유체의 소량의 전사에 도입 될 수있는 오류를 제한 시츄 분비 유체 내 이온 농도?...

<html> 신장 상피 이온 수송은 유기체의 iono- 및 삼투의 기초가. 초파리 melanogaster의 Malpighian (신장) 세관 상피 이온 수송의 분자 메커니즘을 연구 할 수있는 최고의 기회를 제공합니다. 이것은 생리 학적 연구에의 신장 세뇨관의 접근성과 짝을 초파리의 강력한 유전학의 조합 때문이다. 기술 1을 개척 연구자의 ​​이름을 따서 명명 램지 분석, 격리 Malpighian 세관에서 유체 분비 속도를 측정하고, 다우와 동료 2에 의해 1994 년에 초파리 년에 설립되었습니다. 이것은 유체 분비 조절 셀 고유 신호 전달 경로를 정의하는, 초파리 등을 GAL4-UAS 시스템 3,4- 유전 도구를 사용하여 추가 연구를위한 기반을 마련. 예는 많은 다른 6,7 사이에 펩티드 호르몬 (5)에 응답하여 칼슘 신호를 포함한다. 비행 세뇨관의 주요 부분에서 염화칼륨이 풍부한 액체의 분비를 통해 발생에 ve_content &quot;&gt; 유전자 기술 및 고전 생리 학적 연구의 조합 뇨 생성을 도시하고있다.이 주로 양이온 평행 transepithelial 분비 통해 이루어 K는 +뿐만 아니라 나트륨 +는 주체 세포, 및 CL를 통해 -. 성상 세포 8-12 내지 분비 별도로 transepithelial K + 및 나트륨 + 플럭스를 측정하는 능력은 유체 분비의 측정보다 반송기구의 상세한 특성화를 허용 홀로. 예를 들어, 자극되지 초파리 세뇨관에서, 나 +가 / K + -ATPase 억제제 인 ouabain 유체 분비 2에 영향을주지 주체 세포 내로의 흡수량은 유기 음이온 수송 체 억제제 타우로 콜산 (13)에 의해 억제 된 경우에도. 그러나, 린톤 및 오도​​넬은 ouabain과는 탈분극 것을 보여 주었다기저 막 잠재력, 그리고 나 + 플럭스 (9)을 증가시킨다. 대표 결과에 도시 된 바와 같이, 우리는 이러한 발견을 복제하고, 14 병용 감소 K + 플럭스가 있음을 보여 주었다; 증가 나 + 플럭스 및 K 감소 + 플럭스 분비없는 순 변화의 결과로 유체 분비에 반대하는 효과를 가지고있다. . 따라서, &quot;ouabain과 모순&quot;, 즉 두 가지 해결책이 존재하며, 초파리 세뇨관 유체 분비에 영향을주지 ouabain과 초기 관찰 : 제 자극 세뇨관에서 유체 분비 ouabain과 효과는 명백하지 않다 인한 유기 음이온 수송 (13)에 의해 그 흡수; 둘째, 자극되지 세관에서, ouabain과는 (대표 결과와 심판을 참조하십시오. 9) 유체 분비없는 순 변화의 결과로, transepithelial 나 +와 K + 플럭스에 영향을 반대하고있다. 따라서, 나 +의 주요 역할 / K + -ATP비자극 세뇨관에서 ASE는 나트륨 +는 기저 외측 막에 걸쳐 전송 프로세스 -coupled위한 유리한 농도 기울기를 생성하는 세포 나 + 농도를 낮출 수있다. 실제로, 별도로 나 +와 K + 플럭스를 측정하여, 우리는 세관이 비행 나트륨 - 칼륨 - 2 - 염화 송체 (NKCC)를 부족하지 ouabain과 첨가 한 후 더 이상의 감소, transepithelial K + 플럭스 감소했으며, transepithelial의 변화가 있음을 보여 주었다 나 + 14 플럭스. 이러한 연구 결과는 NKCC를 통해 세포를 입력 나 +가 나 + / K + -ATPase를 통해 재활용 우리의 결론을지지했다. 다른 예에서, Ianowski는 외. mM의 10 mM 내지 6 욕 K + 농도를 낮추면 유체 분비없는 순 변화와 Rhodnius의 prolixus에서 세뇨관에서 transepithelial의 K + 플럭스 증가 transepithelial 나 + 플럭스 감소 관찰 <sup&gt; 15. 애벌레 세관에서 나 + 플럭스 및 K + 플럭스에 차등 효과는 염분 (17) 양육에 대한 응답으로 다양한 소금 다이어트 (16)와 두 모기 종에 대한 응답으로 초파리 세관에서 관찰되었다. 램지 분석 준비 transepithelial 이온 플럭스의 측정에서 가장 큰 도전은 분비 된 유체 내 이온 농도의 판정이다. 이 문제는 불꽃 photometery (18), 방사성 이온 (19)의 사용, 전자 프로브 파장 분산 분광기 (20)를 포함하여 다양한 솔루션을 만났다되었습니다. 이러한 기술은 이온 농도의 측정을위한 도구로 분비되는 액체 방울의 전송을 필요로한다. 비자극 초파리 세뇨관 분비 유체의 부피가 작기 때문에, 일반적으로 ~ 0.5 NL / 분, 이것은 기술적 도전을 제기하고 분비 된 유체의 일부가 오류가있는 경우에도 소개이전에 잃었다. 대조적으로, 특정 이온 전극의 사용은 인 시츄 (이온 농도가 계산 될 수있는) 이온 활성의 측정을 허용한다. 현재 프로토콜은 K + 이온 운반체 (21)로 발리 노마 이신 (Valinomycin) 사용 Rhodnius의 세뇨관 걸쳐 transepithelial의 K + 플럭스를 측정하도록 Maddrell 동료가 사용하는 각색 및 또한 4- 급의 -butylcalix [4] 아렌 - 테트라 아세트산의 사용을 설명했다 테트라 에틸 에스테르 계 나은 등 Messerli 특징으로 특이 이온 특정 전극을 +. 등. (22). 이온 특정 전극 성인 9,23과 애벌레 (16) 초파리에서 램지 분석에 Malpighian 세관에 의해 분비되는 유체의 이온 농도를 측정하는 데 사용 된, 뉴질랜드 알파인 웨타 (Hemideina 마오리어) (24) 모기 (17). 여기서는 상세히 램지의 사용 등을 서술유체 분비 초파리 melanogaster의 발 Malpighian 세뇨관의 속도뿐만 아니라 분비 유체 내 K + 및 나트륨 +의 농도를 결정하는 이온 고유의 전극을 사용하므로 transepithelial 이온 플럭스의 계산을 측정하기 말한다. 분석의 개요는도 1에 제공된다. <img alt="그림 1" src="/files/ftp_upload/53144/53144fig1.jpg" /> Malpighian 세관의 그림 1. 도식과 이온 농도를 측정하기 위해 이온 특정 전극의 사용과 램지 분석. 이 그림은 램지 분석에 대한 설정을 보여줍니다. (A) 각 플라이는 네 세뇨관, 전방 세뇨관의 쌍 및 후방 세뇨관의 쌍에 의해 둘러싸인 체액 복강 그 플로트있다. 각 쌍에서 두 세관은 중장 hindgu과의 교차점에 뇨를 비운다 요관에서 합류티. 세관은 블라인드 종료됩니다. 소변 (빨간색으로 표시) 유체 분비 주요 세그먼트에 의해 발생하고, 장에 요관으로 밖으로 흐른다. 절개 후 세관 쌍 요관을 직감에 병행하여 해리된다. (B)은 세뇨관의 쌍은 분석 접시의 웰 내에서 입욕 식염수 방울로 전달된다. 여기에 언급 된 두 세뇨관의 일 &quot;앵커 세관은,&quot;금속 핀 감싸 불활성이다. 다른 세관은 분비의 가느 다란 관이다. 초기 세그먼트 (유체 분비하지 않는)과 분비 세관의 주요 부분은 목욕 식염수의 액적 내에 남아있다. 생체 내에서 발생하는 것에 이온과 목욕 식염수에서 주 세그먼트의 세관 루멘에 물 이동, 그리고, 요관으로 이동합니다. 낮은 세그먼트 (파란색) 목욕 식염수 때문에 불활성 밖에 있습니다. 요관이 절단되기 때문에, 유체는 분비 요관의 절단 단부로부터 액적으로 나온다. 티그 분비는 계속 유체 액적 시간 경과 확대 분비, 그 직경은 접안 마이크로 미터를 사용하여 측정된다. 미네랄 오일 층은 분비 된 유체의 증발을 방지한다. 참조 및 이온 특정 전극이 분비 유체의 이온 농도를 측정한다. <a href="https://www.jove.com/files/ftp_upload/53144/53144fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</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, 30G 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 ionphore 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></td>
		</tr>
		<tr>
			<td>Schneider&#39;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&#176; 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

신장 상피 이온 수송의 변조는 생물체가 외부 환경 변화에 직면 이온과 삼투압 항상성을 유지할 수 있습니다. 초파리 melanogaster의 Malpighian (신장) 가느 다란 관으로 인해이 생물 및 생리 학적 연구에의 신장 세뇨관의 접근성의 강력한 유전학, 상피 이온 수송의 분자 메커니즘을 연구 할 수있는 최고의 기회를 제공합니다. 여기서는 유체 분비 나트륨 및 칼륨 농도를 측정하는 이온 고유의 전극을 이용하여, 절연 플라이 신 세뇨관에서 유체 분비 속도를 측정하기 램지 분석의 사용을 기술한다. 이 분석은 이온 농도를 측정하는 별도의 장치에 분비 된 유체를 전송할 필요없이, 20 시간에 세관의 transepithelial 유체와 이온 플럭스 ~ 연구를 허용한다. 유전자는 별개의 세뇨관 수송 공정에서 특정 유전자의 역할을 평가하기 위해 분석 될 수있다. 또한, Bathing 식염수 첨가 화학적 특성, 또는 약물 또는 호르몬의 효과를 조사하기 위해 수정 될 수있다. 요약하면,이 기술은 이러한 반송기구의 상피 이온 초파리 세뇨관 운송뿐만 아니라, 규정의 기본 메커니즘의 분자 특성화를 허용한다.

도 7 및도 8은 유전 학적 및 약리학 적으로 별개의 K + 및 나트륨 + 플럭스만으로는 유체 분비 속도를 측정함으로써 캡쳐되지 않은 정보를 구분할 수있는 K + 및 나트륨 + 농도를 측정하기 위하여 이온 - 특정 전극 램지 분석의 사용을 보여준다.도를 나트륨 + 플럭스 (도 7b)이 변경되지 않은 상태 NKCC에서 동형 널 돌연변이...

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

이 프로토콜은 초파리에서 격리 Malpighian (신장) 세관에서 유체 분비 속도를 측정 할 수있는 램지 분석의 사용을 설명합니다. 또한, 이온 전극의 특정 사용 transepithelial 이온 플럭스의 계산을 가능하게 분비 유체 나트륨 및 칼륨 농도를 측정하기 위해, 설명한다.

램지 분석의 사용은 유체 분비 및 이온 플럭스 요금을 측정하는<em&gt; 초파리</em&gt; Malpighian하는 Tubule

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

Research

JoVE Journal

Biology

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

11.7K 조회수.  University of Texas Southwestern Medical Center.  이 프로토콜은 초파리에서 격리 Malpighian (신장) 세관에서 유체 분비 속도를 측정 할 수있는 램지 분석의 사용을 설명합니다. 또한, 이온 전극의 특정 사용 transepithelial 이온 플럭스의 계산을 가능하게 분비 유체 나트륨 및 칼륨 농도를 측정하기 위해, 설명한다. 

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

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

의 유사 분열 유학을위한 Microinjection 기법 Drosophila melanogaster Syncytial 엠브료

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

연구

생물학

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

배아 줄기 세포 생활에서 염색질 단백질 역학을 측정하는 Assays (FRAP 및 플립)를 Photobleaching

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

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

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

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

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

인슐린 주입 및 성인에서 인슐린 민감도를 측정하는 Hemolymph 추출 Drosophila melanogaster</em

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

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

의 수명 측정<em&gt; Drosophila melanogaster</em

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

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

사멸 세포 간극의 이미지를하는 동안 라이브<em&gt; 초파리</em&gt; 배아

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

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

의 번데기 케이스를 통해서 영상<em&gt; 초파리</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

정량 면역 형광 분석은 중심체에서 단백질 수준의 변화를 측정하기 위해

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

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.

형광 유전자 인코딩 ph 지시자 초파리 melanogaster 말 관 Epithelia에 광학 정량화의 세포내 pH

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

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

세균성 감염 및 Shedding 초파리 melanogaster 에

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

라이브 드로소필라 멜라노가스터 에그 챔버에서 내인성 mRNA 를 시각화하고 추적

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...