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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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

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

세포 배양과 병아리 배아에서 축슨 유도 수용체의 역학을 평가하기 위해 pHluorin의 사용

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

신경과학

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

TIRFM과 pH 민감성 GFP - 프로브는 SH-SY5Y 신경 모세포종 세포에서 신경 전달 물질 소포 역학을 평가하기 : 세포 이미징 및 데이터 분석

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

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

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

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

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

액포와 세포질의 pH 측정<em&gt; 생체 내</em&gt; 효모 세포 현탁액의

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

생물학

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

공 초점 현미경을 사용하여 살아있는 세포에서 세포 내 이입과 세포질 구획 동시 pH 측정

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

트리파노소마 브루세이(Trypanosoma brucei)의 글리코솜 pH를 위한 pHluorin2 기반 센서 개발

Measuring Dynamic Glycosomal pH Changes in Living Trypanosoma brucei

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. Little is known about the cellular responses generated when environmental glucose levels change. In both bloodstream and procyclic form (insect stage) parasites, glycosomes house most of glycolysis. These organelles are rapidly acidified in response to glucose deprivation, which likely results in the allosteric regulation of glycolytic enzymes such as hexokinase. In previous work, localizing the chemical probe used to make pH measurements was challenging, limiting its utility in other applications.
This paper describes the development and use of parasites that express glycosomally localized pHluorin2, a heritable protein pH biosensor. pHluorin2 is a ratiometric pHluorin variant that displays a pH (acid)-dependent decrease in excitation at 395 nm while simultaneously yielding an increase in excitation at 475 nm. Transgenic parasites were generated by cloning the pHluorin2 open reading frame into the trypanosome expression vector pLEW100v5, enabling inducible protein expression in either lifecycle stage. Immunofluorescence was used to confirm the glycosomal localization of the pHluorin2 biosensor, comparing the localization of the biosensor to the glycosomal resident protein aldolase. The sensor responsiveness was calibrated at differing pH levels by incubating cells in a series of buffers that ranged in pH from 4 to 8, an approach we have previously used to calibrate a fluorescein-based pH sensor. We then measured pHluorin2 fluorescence at 405 nm and 488 nm using flow cytometry to determine glycosomal pH. We validated the performance of the live transgenic pHluorin2-expressing parasites, monitoring pH over time in response to glucose deprivation, a known trigger of glycosomal acidification in PF parasites. This tool has a range of potential applications, including potentially being used in high-throughput drug screening. Beyond glycosomal pH, the sensor could be adapted to other organelles or used in other trypanosomatids to understand pH dynamics&#160;in the live cell setting.

저자 스포트라이트: pHluorin2 Biosensor를 사용한 Trypanosoma brucei 의 당체 pH 모니터링의 발전

We describe a method to study how pH responds to environmental cues in the glycosomes of the bloodstream form of African trypanosomes. This approach involves a pH-sensitive heritable protein sensor in combination with flow cytometry to measure pH dynamics, both as a time-course assay and in a high-throughput screen format.

살아있는 트리파노소마 브루세이(Trypanosoma brucei)의 동적 당염색체 pH 변화 측정

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. ...

면역학 및 감염병학

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 ...

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.

성인 Drosophila 창자에 있는 창자 산성화를 감시합니다

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

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

세포 배양과 병아리 배아에서 축슨 유도 수용체의 역학을 평가하기 위해 pHluorin의 사용

TIRFM과 pH 민감성 GFP - 프로브는 SH-SY5Y 신경 모세포종 세포에서 신경 전달 물질 소포 역학을 평가하기 : 세포 이미징 및 데이터 분석

액포와 세포질의 pH 측정

공 초점 현미경을 사용하여 살아있는 세포에서 세포 내 이입과 세포질 구획 동시 pH 측정

살아있는 트리파노소마 브루세이(Trypanosoma brucei)의 동적 당염색체 pH 변화 측정

살아있는 트리파노소마 브루세이(Trypanosoma brucei)의 동적 당염색체 pH 변화 측정

살아있는 트리파노소마 브루세이(Trypanosoma brucei)의 동적 당염색체 pH 변화 측정

램지 분석의 사용은 유체 분비 및 이온 플럭스 요금을 측정하는

성인 Drosophila 창자에 있는 창자 산성화를 감시합니다