이 작품은 NIH DK092408에 의해 지원 되었다 그리고 MFR. AJR에 DK100227 T32-DK007013에 의해 지원 되었다. 저자 CapaR GAL4와 c724 GAL4 박사 줄리안 A.T. 다우 감사 하고자 초파리 주식. 우리는 또한 실험적인 비행 십자가 유지 하는 지원에 대 한 야 곱 B. 앤더슨을 감사 합니다.

저자는 공개 없다.

Drosophila MTs에 pH나 의 정량화의 성공을 전적으로 추출 된 MTs와 장착 및 해 부그림 A - ( C)의 품질의 상태에 따라 달라 집니다. 따라서, 조직으로의 주의 깊은 취급 설명은 필수적입니다. PLL에 갓 코팅 슬라이드 실질적으로 솔루션 이전 노출 슬라이드 원조 마운트 장착 그들 보다 훨씬 더 많은 접착제 경향이. 주의 설치 또한 뚜렷한 산 세그먼트 (<s...

이 프로토콜의 목표는 정량화의 세포내 pH (pHi)는 유전자 인코딩 pH-표시기 (GEpHI)를 사용 하 여 설명 하 고 어떻게 모델 곤충 (디의에서 basolateral H+ 교통을 평가 하기 위해이 메서드를 사용할 수 있습니다 설명 melanogaster) 신장 구조, 말 관 (MT). MTs 과일 파리의 배설 기관으로 봉사 하 고 몇 가지 주요 측면1포유류 nephron 기능적으로 비슷합니다. MTs는 흉부와 복 부의 tubules (앞쪽에 및 사후)의 2 쌍으로 정렬 됩니다. 각 산의 단일 셀 상피 튜브 metabolically 활성 주 셀의 독특한 꼭대기 구성 (luminal)과 basolateral (hemocoel) 극성으로 삽입된 방사상 세포. 형태학 상으로, 기능적으로, 앞쪽 MTs 3의 구성 그리고 발달 별개 세그먼트, 특히 초기 크기 세그먼트, 전환 세그먼트, 및2ureter 조인 분 비 주요 세그먼트. 셀룰러 규모 루멘으로 트랜스 상피 이온 수송 basolateral 나+-K+뿐만 아니라 꼭대기 원형질 막 V ATPase3 와 알칼리-금속/H+ 교환기에 의해 수행 됩니다-ATPase4, 내부 정류기 K+ 채널5, 나+-구동 Cl−/HCO3− 교환기 (NDAE1)6및 Na+-K+-2 Cl− cotransporter (NKCC; Ncc69)7, 방사상 세포 중재 Cl- 물8,9전송 하는 동안. 이 복잡 하지만 액세스할 수 physiologic 시스템 초파리의 유전 및 행동 다양 한 도구와 함께 내 생 이온 전송 메커니즘의 조사를 위한 우수한 기회를 제공 합니다.
이 프로토콜에 대 한 근거는 행동과 다른 모델 시스템 도구의 수출을 셀에서 통합에 대 한 잠재력을 가진 상피 이온 수송을 공부에 대 한 유전자가 단 시스템을 설명 했다. PHerry10의 표현, 녹색 pH에 민감한 슈퍼 황도 pHluorin11,12 (SEpH) 및 빨간색 pH을 구분 하지 않는 mCherry13, MTs의 융합에서 파생 된 GEpHI 허용에 H+ 전송의 정량화 높은 K+/nigericin 교정 기술14를 통해 단일 산 세포. 마찬가지로 많은 이온 운송업 자 H+ 해당 이동, 세포내 pH나 의 정량화 전송기의 다양 한 통해 이온 이동의 기능적 표현으로 제공 합니다. 또한 조직의 특정 transgene15 에 RNA 간섭 (RNAi)16 식 세포 이미징 및 전체-기관 분석17 와 결합 될 수 있는 강력한 유전자 도구를 제공 하는 초파리 MT 모델 시스템 , 18 , 19 관 기능 동작에 분자에서 수직 통합 된 강력한 도구 집합을 만들 수의. 이 스탠드 반면 다른 많은 프로토콜으로 역사적으로 그러한 측정 의존 해야 복잡 한 상피 생물학을 평가 하 고 마이크로 해 부, 정교한 이온 선택적인 전극20,21, 발굴 그리고 비싼 pH에 민감한 염료22 제한적인 로드 요구와 다른 유형의 조직에 가난한 세포 특이성. GEpHIs는 광범위 하 게 pH나 다양 한 세포 유형23을 측정 하기 위해 사용 되어 왔습니다. 초기 작품 고유의 pH 감도의 녹색 형광 단백질 (GFP) pH나 배양된 상피 세포24 을 모니터링 하는 데 악용 하지만 지난 2 년간 본 균27 뉴런25명과26, 사용 GEpHIs , 그리고 식물 세포28. 세포질 GAL4/UAS 식 시스템15 와 초파리 MT의 생리 적인 접근을 통해 유전 구성의 대상에 대 한 잠재력의 조합을이 pH의 조사에 대 한 이상적인 준비 하 게 나 레 귤 레이 션 및 상피 이온 수송.
pH나 규제 수십 년 동안 공부 되었습니다 하 고 생활에 필수적 이다. MT 준비는 강력한 모델 pH나 규제의 생리학을가 르 치지만 또한 수행을 정교한의 pH나 레 귤 레이 션 ex vivo에서 비보조사를 제공 합니다. 이 프로토콜 초파리 NH4Cl 펄스 산 기술21, 로드를 사용 하 여 산의 상피 세포의 basolateral 막에 걸쳐 H+ 움직임의 정량화를 설명 하지만 pH 지시자로 유전자 인코딩, 의무가 transgenesis 및 라이브 이미징 모든 준비를 이러한 메서드 및 그들의 이론적인 틀을 적용할 수 있습니다.

<table><tbody><tr><td>Poly-L-Lysine (PLL) Solution</td><td>Sigma-Aldrich</td><td>P4832</td><td>Store at 4 &amp;deg;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 &amp;deg;C.</td></tr><tr><td>Adhesive Perfusion Chamber Covers, adhesive size 1 mm, chamber diameter &amp;times; thickness 9 mm &amp;times; 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&#039;s Medium</td><td>Fisher Scientific</td><td>21720024</td><td>Store at 4 &amp;deg;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 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&quot;, OD 1/8&quot;, thickness 1/32&quot;</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 &lt;em&gt;Drosophila melagnogaster&lt;/em&gt;</td><td>Available from Romero Lab</td><td></td><td>First published: Citation 10</td></tr><tr><td>capaR-GAL4 driver line &lt;em&gt;Drosophila melagnogaster&lt;/em&gt;</td><td>Available from Romero Lab</td><td></td><td>First published: Citation 32</td></tr><tr><td>c724-GAL4 driver line &lt;em&gt;Drosophila melagnogaster&lt;/em&gt;</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 &amp;#956;L 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

상피 이온 수송 체계 이온 항상성 뿐만 아니라 필수적인 세포 전기 화학 기온 변화도의 유지 보수에 필수적입니다. 세포내 pH (pH나)은 많은 이온 운송업 자에 의해 영향을 하 고 따라서 pH나 모니터링은 운송업 자 활동을 평가 하기 위한 유용한 도구. 현대 유전자 인코딩 pH-지표 (GEpHIs) 세포 및 subcellular 규모 pH나 그대로 셀에의 부 광 량을 제공합니다. 이 프로토콜 설명 세포질 pH나 규제의 초파리 melanogaster 의 말 Tubules (MTs)에서 ex vivo 라이브 이미징 통해 pHerry, pK의사 비율 GEpHI의 실시간 정량화를 는 cytosol에서 pH 변화 추적에 적합. 추출 된 성인 비행 MTs 단일 셀 레이어 epithelia의 형태학 상으로 그리고 기능상 다른 섹션으로 구성 되며 상피 전송의 조사를 위해 접근 가능 하 고 유전자 세공 모델으로 사용할 수 있습니다. GEpHIs 기존 pH에 민감한 형광 염료와 이온 선택적 전극 위에 여러 가지 이점을 제공합니다. 적절 한 모터 요소 사용할 수 있습니다 제공 GEpHIs 뚜렷한 세포 인구 레이블을 수 있습니다. 이 라벨은 전 비보 vivo에서, 그리고 제자리에 , 준비는 본질적으로 다른 유형의에 특히 유용 합니다. GEpHIs 또한 반복된 염료 치료 또는 조직의 외부화에 대 한 필요 없이 시간이 지남에 그대로 조직에 pHi 의 정량화를 허용합니다. 현재 GEpHIs의 주요 단점은 경향을 cytosolic 포함 조직 손상에 대 한 응답에서에서 집계-표현식을 구성 합니다. 이러한 단점, 그들의 솔루션 및 GEpHIs의 고유의 장점은 추출 된 MTs의 기능적으로 명료한 원금과 방사상 세포의 basolateral 양성자 (H+) 교통의 평가 통해이 프로토콜에서 설명 했다. 기술과 분석 설명 다양 한 척추 동물 및 무척 추 동물 준비에 쉽게 적응할 수 있다, 분석 결과의 세련 특정 전송기를 통해 이온 플럭스의 복잡 한 결정을 연구소 교육에서 조절 될 수 있습니다.

건강 한 조직 및 앞쪽 MTs의 적절 한 식별이이 프로토콜의 성공에 생명 이다. 해 부, 동안 주의 해야 직접 터치 하는 MTs와만 핸들을 그들은 MTs를 직접 잡지로 ureter 파손 (그림 4A- B)로 이어질 것입니다. MTs는 플랫 슬라이드에 휩 쓸, tubules는 가능한 조금만 진 해야 합니다 고이 피할 초과 모션 단일 셀 상피 층 (그...

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

세포 이온 수송 종종 세포내 pH를 모니터링 하 여 평가 될 수 있다 (pH나). 유전자 Encoded pH-지표 (GEpHIs) 그대로 셀에 세포내 pH의 부 광 량을 제공합니다. 이 프로토콜 내용을 통해 셀룰러 ex vivo 라이브 이미징 pHerry와 초파리 melanogaster 의 말 tubules의 세포내 pH의 정량화, 의사 비율 유전자 인코딩 pH 지시자.

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 ...

optical-quantification-intracellular-ph-drosophila-melanogaster

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Biology

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

10.1K 조회수.  Mayo Clinic College of Medicine. 세포 이온 수송 종종 세포내 pH를 모니터링 하 여 평가 될 수 있다 (pH나). 유전자 Encoded pH-지표 (GEpHIs) 그대로 셀에 세포내 pH의 부 광 량을 제공합니다. 이 프로토콜 내용을 통해 셀룰러 ex vivo 라이브 이미징 pHerry와 초파리 melanogaster 의 말 tubules의 세포내 p...

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

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

라이브 초파리 난소 조직에 여 포 줄기 세포 계보의 이미징 세포내 pH에 대 한 방법

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

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발생학

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

에 대한 Mesoscopic 형광 Tomography 인 - 생체내</em개발> 이미징 Drosophila</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

생물학

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.

의 생리적 반응을 측정<em&gt; 초파리</em&gt; 라이브 칼슘 이미징을 사용하여 지질 페로몬에 감각 뉴런

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

면역학 및 감염병학

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

전 세포 전압 클램프 기록을위한 전기 생리 학적 방법<em&gt; Drosophila</em&gt; 광 수용체

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

신경과학

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

과일 파리, 초파리 melanogaster에 비보 먹어서 분석 결과 사용 하 여의 세포 면역 반응 평가

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

ImageJ를 사용 하 여 붙일 태그가 단백질의 편견된 분석 통해 초파리 에서 Neurodegeneration의 양적 세포 생물학

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

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

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The Drosophila&#160;melanogaster Malpighian (renal) tubule offers an unparalleled opportunity to study the molecular mechanisms of epithelial ion transport, due to the powerful genetics of this organism and the accessibility of its renal tubules to physiological study. Here, we describe the use of the Ramsay assay to measure fluid secretion rates from isolated fly renal tubules, with the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid. This assay allows study of transepithelial fluid and ion fluxes of ~20 tubules at a time, without the need to transfer the secreted fluid to a separate apparatus to measure ion concentrations. Genetically distinct tubules can be analyzed to assess the role of specific genes in transport processes. Additionally, the bathing saline can be modified to examine the effects of its chemical characteristics, or drugs or hormones added. In summary, this technique allows the molecular characterization of basic mechanisms of epithelial ion transport in the Drosophila tubule, as well as regulation of these transport mechanisms.

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

This protocol describes the use of the Ramsay assay to measure fluid secretion rates from isolated Malpighian (renal) tubules from Drosophila melanogaster. In addition, the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid, allowing calculation of transepithelial ion flux, is described.

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 ...

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins

Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally important for cell function and cell integrity of neurons. Drosophila photoreceptor cells have become a model for studying membrane protein trafficking. Besides rhodopsin, which upon illumination becomes internalized from the photoreceptor membrane and is degraded, the transient receptor potential-like (TRPL) ion channel in Drosophila exhibits a light-dependent translocation between the rhabdomeral photoreceptor membrane (where it is located in the dark) and the photoreceptor cell body (to which it is transported upon illumination). This intracellular transport of TRPL can be studied in a simple and non-invasive way by expressing eGFP-tagged TRPL in photoreceptor cells. The eGFP fluorescence can then be observed either in the deep pseudopupil or by water immersion microscopy. These methods allow detection of fluorescence in the intact eye and are therefore useful for high-throughput assays and genetic screens for Drosophila mutants defective in TRPL translocation. Here, the preparation of flies, the microscopic techniques, as well as quantification methods used to study this light-triggered translocation of TRPL are explained in detail. These methods can be applied also for trafficking studies on other Drosophila photoreceptor proteins, for example, rhodopsin. In addition, by using eGFP-tagged rhabdomeral proteins, these methods can be used to assess the degeneration of photoreceptor cells.

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins

Here, non-invasive methods are described for localization of photoreceptor membrane proteins and assessment of retinal degeneration in the Drosophila compound eye using eGFP fluorescence.

eGFP 태그 단백질을 사용하여 초파리 광수용체 세포에서 막 단백질 밀매를 연구하기

Membrane protein trafficking regulates the incorporation and removal of receptors and ion channels into the plasma membrane. This process is fundamentally ...

Malpighian Tubule Dissection from an Adult Fly: Isolating an Osmoregulatory and Excretory Organ

Source: Rossano, A. J. and Romero, M. F. <a href="https://www.jove.com/video/55698/" target="_blank">Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator</a>. J. Vis. Exp. (2017).
This article describes the dissection of the anterior Malpighian tubules (MT) from a female Drosophila fly and an example protocol from Rossano et al. (2017) in which the MTs are prepared for live imaging in a perfusion chamber.

성충 파리의 Malpighian Tubule Dissection: Isolating an Ossmoregulatory and Excretory Organ(성충 파리의 Malpighian Tubule Dissection: Isolating an Osmoregulatory and Excretory Organ)

Source: Rossano, A. J. and Romero, M. F. Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a ...

JoVE Encyclopedia of Experiments

Drosophila melanogaster (fruit fly)

Adult

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells

<a href='https://app.jove.com/v/63375/studying-membrane-protein-trafficking-drosophila-photoreceptor-cells'>Source: Wagner, K. et al., Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells Using eGFP-Tagged Proteins.&#160;J. Vis. Exp. (2022).</a>This video demonstrates the visualization of membrane protein trafficking in Drosophila photoreceptor cells using eGFP-tagged TRPL proteins. It details the preparation of transgenic flies for imaging, the capture of fluorescence images of photoreceptor cells, and the quantification of TRPL trafficking by measuring relative eGFP fluorescence in the rhabdomeres.

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

요약

더 많은 비디오 탐색

이 비디오의 챕터

라이브 초파리 난소 조직에 여 포 줄기 세포 계보의 이미징 세포내 pH에 대 한 방법

에 대한 Mesoscopic 형광 Tomography 인 - 생체내 이미징 Drosophila

의 생리적 반응을 측정

전 세포 전압 클램프 기록을위한 전기 생리 학적 방법

과일 파리, 초파리 melanogaster에 비보 먹어서 분석 결과 사용 하 여의 세포 면역 반응 평가

ImageJ를 사용 하 여 붙일 태그가 단백질의 편견된 분석 통해 초파리 에서 Neurodegeneration의 양적 세포 생물학

램지 분석의 사용은 유체 분비 및 이온 플럭스 요금을 측정하는

eGFP 태그 단백질을 사용하여 초파리 광수용체 세포에서 막 단백질 밀매를 연구하기

성충 파리의 Malpighian Tubule Dissection: Isolating an Ossmoregulatory and Excretory Organ(성충 파리의 Malpighian Tubule Dissection: Isolating an Osmoregulatory and Excretory Organ)

Studying Membrane Protein Trafficking in Drosophila Photoreceptor Cells