Este trabalho foi financiado pelo NIH R01 GM50322 a PM Kane. Os autores agradecem ao Dr. Rajini Rao, Johns Hopkins University para fornecer as leveduras pHluorin plasmídeos e conselhos sobre as medições de pH raciométrica e Dr. Gloria A. Martinez Munoz para trabalhar fora estes protocolos para o nosso laboratório.

1. Medição do valor pH vacuolar Em a Vivo Usando BCECF-Rádio AM <ol><li> Cresça um de 50 ml de cultura líquido de o estirpe de levedura para ser medido em a médio o desejar o pernoite. A meta é para têm células de em fase de meados-de log (DO600 (densidade óptica a 600 de nm) à mensuração dos aproximadamente, de 0,8 para a suspensão). </li><li> Da pelota as células de levedura, por centrifugação em. Ressuspender o o pellet em 0,6 ml de o meio de crescimento e transferência para um tubo de microcentrífuga de que tenha sido, previamente tarado com. De pellets as células novamente em um de microcentrífuga de a 2.000 xg por 60 sec. Remover filtro-se o sobrenadante, tão completamente quanto possível e em seguida, pesam o pellet célula. Ressuspender o pelete até uma densidade final de 0,5 de g / ml de (w / v) de; uma cultura de este volume irá dar um pelete de células de, aproximadamente, de 200 mg, e 200 jil de tampão de iria ser adicionado a dar um volume final de de 400 uL. </li><li> Adicionar BCECF-AM às o suspensão celular a uma operação de concentração final de 50 mM de a partir de um Estoque 12 de mM de preparado no seio de DMSO. Misture bem e, em seguida, incUbate as células em 30 de ° C durante de automóvel 30 min. em uma plataforma de de balanço ou o tambor de rolete. </li><li> Enquanto as células de estiverem incubando, prepare-se buffers de de calibração. O buffer de de calibração contém 50 mM de MES (2 - (N-morf olino) Solução de ácido etanossulfónico), a 50 mM de HEPES (4 - (2-de hidroxietilo)-1-piperazinoetanossulfónico ácido), de KCl equivalentes a 50 mM, NaCI a 50 mM, do acetato de de amónio 0,2 M, 10 azida de mM de sódio, e 10 mM 2-deoxyglucose, e é ajustado para o pH valoriza apropriado para o seu faixa de calibração com NaOH ou HCl. (Cuidado: A azida de Sódio é altamente tóxico, e deverá ser manuseado com cuidado.) Para a medição da de de pH em células de-Tipo de deixa selvagens, nós iria preparar vários misturas de calibração a pH 5,5 a 6,5, mas para os mutantes prevê venham a ter um pH vacuolar obter mais alcalina (de mutants vma), nós iria preparar buffers de pH adicionais, mais elevados. </li><li> Aliquotar de 2 ml de tampão de de calibração para cada valor de pH em 15 ml tubos cónicos de, e de adicioná monensina na dieta (Estoque 15 de mM) e nigericina (Estoque 2 mM de) para dar origem concentrações finais de 110 | iM monensina de e 15 º uM nigericina, r respectively. Misture bem on um misturador de vórtice. (Caution: Both monensina e nigericina são tóxicos e deve ser manuseado com cuidado.) </li><li> Quando o tempo de incubação BCECF-Rádio AM é concluída, peletizar a suspensão de células por centrifugação durante 30 de seg Rácio às 2,000 xg, em um de microcentrífuga de. Ressuspender as células é de 1 ml de meio de crescimento carente de glicose e centrifuga-se como acima. Repita este passo de lavagem com uma vez, e, em seguida, fim de ressuspender a sedimento em 200 jil de meio de crescimento sem glicose. Lugar on de gelo. </li><li> Juntar 20 ul de a suspensão de células a cada tubo de de calibração de pH preparado acima. Incubar a uma temperatura 30 de ° C durante 30-60 mín. sobre um tambor rolete. </li><li> Defina o fluorímetro para medir alternadamente em comprimentos de onda de excitação 450 nm e 490 nM, ambos os com um comprimento de onda de emissão de de 535 nm. Defina a temperatura do câmara de amostra para de 30 ° C. Perform todas as medições de, com agitação contínuas de-se a mistura na cuvete de. </li><li> Adicionar 1,96 ml de do EEM dentro em 1 mM de (ajustado-se até pH 5 ou 7, dependendo da o pH de meio de crescimento as &#39;células que umand o design experimental). Juntar 20 jil de suspensão de células na cuvete de. Initiate medições de fluorescência. Nós coletamos tanto kinetic contínua e de dados em tempo-ponto em diferentes experimentos. Para obter dados cinéticos contínuas, nós tomamos medições a cada 6 seg Rácio para de automóvel 5 min,, em seguida, de adicioná glicose para uma operação de concentração definitivo de glicose de 50 mM de, e continuar medição de durante 5-10 min. Para medições de solteiras time-de ponto, nós geralmente efectuar medições em 1 e 5 min. depois de adição de células ao o cuvete, as de adicioná a glicose como em as medições cinéticos, e outra em seguida, tomar-se outra medição de automóvel 5 min. após a adição da glucose. Estas adições pode ser variada, e de componentes do adicionais de (inibidores da, etc) também pode ser adicionado. </li><li> Depois que os medições experimentais são concluída, remova os tubos de de calibração a partir de o 30 ° C de incubação e transferir toda a ml volume de 2 para cada um para o cuvette fluorímetro. Meça fluorescência em as mesmas configurações de (ver 1.8) a cada 5 sec ao longo de um total de 30 de sec para cada amostra. </li><li>Exportar dados de fluorescência para Microsoft Excel. (Para o nosso fluorímetro, isto requer exportação de dados como texto em forma de tab-delimited e importação para Excel.) Obter um curva de calibração por meio do cálculo a proporção de de fluorescência a 490 nm para 450 nm para cada mistura de calibração. O rácio de fluorescência é, em seguida, plotados vs de pH para obter um curva de calibração. </li><li> Converta os dados de experimentais para rácio de de fluorescência e calcular do pH utilizando um a curva padrão. De pH vacuolar pode, em seguida, plotados contra o tempo (cinética da sua as alterações de pH) ou comparado ao abrigo diferentes condições (Figura 1) ou em diferentes estirpes mutantes. </li></ol> 2. Medição do valor pH Cytosolic Em a Vivo Usando pHluorin Levedura <ol><li> Transform o estirpe de levedura o desejar com o pHluorin plasmídeo de levedura por protocolos de forfetários, e selecionar para transf ormantes sobre suplementado meio mínimo de uracilo faltando (em SC-de uracil),. </li><li> Cresça um de 50 ml de cultura líquido de células transformadas especialmente em SC-de uracilo até à fase meados-de log (Posição OD <sub&gt; 600 = 0,8 ou inferior)-. </li><li> Prepare a padrões de calibração de como se descreveu para de pH vacuolar, mas buffer para uma gama de pH apropriado para as medições de pH citosólicas, geralmente de pH 6-8. Aliquotar de 2 ml de tampão de de calibração ajustado para o pH o desejar em várias de 15 ml tubos cónicos de. Adicionar monensina e do nigericina tal como descrito de cima (1,4) e nos misture bem. </li><li> Colha as células por centrifugação, tal como descrito, supra, e fim de ressuspender o sedimento em 600 ul de o meio de crescimento. Transfira para uma tubo de microcentrífuga de pesava, e as células da pelota por centrifugação a 5.000 rpm durante 30 de seg Rácio. Ressuspender o sedimento em 1 ml de meio de crescimento sem glicose (apenas SC-uracilo, cloridrato de-glucose), as células da pelota novamente e repita os. Depois que o centrifugação final, retire-se o sobrenadante tão completamente quanto possível e pesar o pelete célula. Ressuspender com células até uma densidade final de 0,5 de g / ml de em SC-de uracilo com nenhuma da glucose. </li><li> Juntar 20 jil de suspensão celular aos cada tubo de de tampão de de calibração e misture bem sobre um mixador de de vórtice. Incubar a 30 ° C num rotor de tambor giratório durante 60 minutos. </li><li> Configure o fluorímetro para a excitação em comprimentos de onda 405 e 485 nm e um comprimento de onda de emissão de 508 nm. Prosseguir com um único ponto de tempo ou medições cinéticas e adição de glicose como acima descrito para a medição de BCECF. </li><li> Medir a fluorescência das amostras de calibração e construir uma curva de calibração como para BCECF (veja 1,10-1,11). A trama da relação de fluorescência versus pH é linear em toda a gama da maioria das medições de pH citosólicas (pH 6,0-8,0) 9, as medições relação fluorescência tão experimentais são facilmente convertidos em pH. </li></ol>

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Os autores não têm conflitos de interesse de divulgar.

Nós utilizamos esses protocolos para abordar uma série de aspectos da homeostase do pH. Por exemplo, nós compararam respostas citosólicas e pH de células mutantes do tipo selvagem e V-ATPase deficiente 4,5. Examinamos, também, os efeitos das condições de crescimento alterados, pH particularmente extracelular, em resposta pH vacuolar de glicose 3. É importante ressaltar que as respostas que observamos são consistentes com outros métodos de medição de pH quantitativa e com dados bioquím...

a homeostase de pH é uma processo dinâmico e altamente regulamentado em todos os organismos 1,2. Processos bioquímicos são rigidamente regulada pelo pH, e ambientes de intracelulares estão sintonizados para gamas de pH estreitas para permitir que uma actividade óptima de as enzimas de residentes. No entanto, a homeostase pH intracelular pode ser desafiado por mudanças rápidas na pH ambiental, turnos de metabólicas, e de certas vias de sinalização. Além disso, o pH de intracellular pode em si mesmo servir como um sinal importante. Finalmente, muitas organelas manter os valores de de pH lumenal que são distintas a partir de o citosol circundante e essencial para as funções específicas-organela. Os de levedura de Saccharomyces partes cerevisiae um certo número de mecanismos de da homeostase de pH com a eucariotas superiores 2. In as organelas ácidas de o pathway endocytic / lysosomal, pH é controlado, principalmente, pela altamente conservada vacuolar ATPase próton-translocating (V-ATPase), atuando em tandem com muitos exchaniurtas dependentes relativa à gradiente de pH. Todos os células eucarióticas também têm mecanismos de exportação de prótons. In é acreditado fungos e plantas, uma segunda da bomba de próton, distinto na membrana plasma, Pma1, as exportações prótons metabólicas e para ser o dos principais determinantes da pH citosólica e potencial de membrana plasma. A flexibilidade genética de S. cerevisiae e sua importância comercial, fizeram-lhe um modelo de muito interessante e importante para o estudo de a homeostase pH 2. In Além de ser os drivers de primárias de organelle a acidificação, V-ATPases são altamente regulamentados enzimas e nosso laboratório de está interessado em compreender mecanismos de regulação V-ATPase. Rumo a este objetivo, temos vindo a utilizar em medições de pH in vivo de pH vacuolar e citosólica: 1) para monitorar respostas aos mudanças nas condições de extracelulares, tais como privação glicose e readdition, 2 de) para examinar os efeitos de mutações que compromisso de atividade V-ATPase, e 3) para explorar a coordenação de organelle e plasma de membrana bombas de protões 3-5. Estes experimentos só se tornou possível através de o desenvolvimento de robustas indicadores de pH raciométrica passíveis da para usar em células de levedura. . Vegetal et ai mostrou pela primeira vez de que BCECF (2&#39;7&#39;-Bis-(2-carboxietilo) -5 - (e 6 de)-carboxif luoresceina), o qual tem sido utilizado amplamente para medir o pH citosólica em células de mamíferos, se acumula em o vacúolo de levedura em vez de o citosol 6. Esta diferença na BCECF localisation tem sido atribuída a as muitas enzimas hidrolíticas em o vacúolo, os quais são provável responsável pela clivagem do o éster de meti Io acetoxi a partir de BCECF-Rádio AM (éster acetoximetilo de BCECF) e retenção de vacuolar 6. Ali et ai. 7 ainda mais desenvolvido pela medição do pH vacuolar usando BCECF e adaptado estas medições para um formato de leitor de placas de de fluorescência. De Brett et ai. Apresentar-pHluorin de levedura como um meio de de medição de pH citosólica em levedura por expressar os um r r plasmídeo de-borneatiometric a GFP de pH-sensível 8, sob controlo de um promotor específico-levedura 9. O Os espectros de de excitação de tanto BCECF e levedura de cerveja pHluorin são sensíveis-se até pH, assim, elas são usados ​​como indicadores de pH raciométrica na qual o rácio de de de fluorescência a dois comprimentos de onda de excitação, medidos em condições um único comprimento de onda de emissão de, e que providencia uma medida de de pH 8,10. Estes sensores de pH vacuolares e citosólica levedura têm sido utilizados para ambas as medições single-cell and-baseada em população. Medições de-de células Um único 6,11 são executadas por microscopia de de fluorescência e de análise de imagem. De fluorescência vacuolar ou citosólica aqui as dois comprimentos de onda é medido para as cada célula. As medições-baseados em população são efectuado tanto na um leitor de microplacas de com capacidades de de fluorescência adequadas ou o em um f luorimetro. Nós têm, em geral feito os nossos de medições sobre uma fluorímetro, com um porque ele fornece do fácil acesso para adição de componentes, tais como glucose durante engodomedições de cinéticos contínuo. Nossos protocolos de actuais do laboratório para a medição de de de pH vacuolar e citosólica são coletados abaixo; ambos também são facilmente adaptados-se a ensaios de microplacas.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Spectrofluorometer</td>
 <td>Horiba Jobin Yvon</td>
 <td>Model Fluoromax-4</td>
 <td>Temperature control and stirring capability are desirable.</td>
 </tr>
 <tr>
 <td>BCECF-AM</td>
 <td>Invitrogen/Molecular Probes</td>
 <td>B1150</td>
 <td>Prepare a 12 mM stock in dry DMSO, store as aliquots at -20 &deg;C </td>
 </tr>
 <tr>
 <td>monensin</td>
 <td>Sigma</td>
 <td>M5273</td>
 <td>Toxic. </td>
 </tr>
 <tr>
 <td>nigericin</td>
 <td>Sigma</td>
 <td>N7143</td>
 <td>Toxic. </td>
 </tr>
 <tr>
 <td>MES</td>
 <td>Sigma</td>
 <td>M8250</td>
 <td></td>
 </tr>
 </tbody>
</table>

measurement of vacuolar and cytosolic ph in vivo in yeast cell suspensions

Vacuolar e pH citosólica são altamente regulamentados em células de levedura e ocupam uma papel central na homeostase do global de pH. Nós descrever protocolos de para a medição de tipo raciométrico de de pH in vivo utilizando fluoróforos-sensíveis ao pH localizadas para o o vacúolo ou citosol. De pH vacuolar é medido usando BCECF, o qual localizes para o vacúolo em levedura quando introduzido em células de, na sua forma éster de acetoximetilo. De pH Cytosolic é medido com um a GFP de pH-sensível expressou ao abrigo o controle de um promotor de levedura, de levedura pHluorin. Métodos para a medição de dos rácios de de fluorescência em suspensões de celulares de leveduras em um fluorímetro são descritos. Através de estes protocolos, as medições de pontos tempo de solteiro de de pH ao abrigo condições diferentes ou em diferentes mutantes de de levedura tenham sido comparados e mudanças no pH ao longo do tempo tenham sido monitorizados. Esses métodos também tiverem sido adaptadas ao um formato de leitor de placas de a fluorescência para a experimentos high-throughput. Vantagens de medições de pH raciométrica mais de outro apdagens atualmente em uso, potenciais problemas de e soluções experimentais, e prospects para uso futuro de estas técnicas também são descritas.

A Figura 1 apresenta os dados de pH vacuolar obtidos em células de levedura de tipo selvagem cultivadas em meio rico (extracto de levedura-peptona-dextrano; YEPD), tamponadas a pH 5 com 50 mM de MES. Frequentemente crescer as células em meio tamponado, porque o pH do meio pode mudar dramaticamente durante o crescimento durante a noite, em especial para um meio mínimo, e verificou-se que o pH do meio de crescimento pode afectar as respostas do pH vacuolar 3. No entanto, também é aceitáve...

Watch this Scientific Journal Video about Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions at JoVE.com

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

PH vacuolar e citosólica pode ser medido em leveduras vivas (<em&gt; S. cerevisiae</em&gt;) As células utilizando corantes fluorescentes raciométrica localizadas em compartimentos celulares específicos. Nós descrevemos processos de medição de pH vacuolar com BCECF-AM, que se localiza no vacúolo em levedura e pH citosólico com uma GFP sensível ao pH raciométrica citosólica (levedura pHluorin).

Medição do valor pH vacuolar e Cytosolic<em&gt; In Vivo</em&gt; Em Suspensions célula de levedura

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

measurement-vacuolar-cytosolic-ph-vivo-yeast-cell

Research

JoVE Journal

Biology

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

19.8K visualizações.  SUNY Upstate Medical University. PH vacuolar e citosólica pode ser medido em leveduras vivas ( S. cerevisiae) As células utilizando corantes fluorescentes raciométrica localizadas em compartimentos celulares específicos. Nós descrevemos processos de medição de pH vacuolar com BCECF-AM, que se localiza no vacúolo em levedura e pH citosólico com uma GFP sensível ao pH raciométrica citosólica (levedura pHluorin).

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

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

Medição in vivo Micro-circulação no músculo esquelético por Intra-vital Microscopia

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Biologia

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Medição quantitativa de invadopodia mediada proteólise da matriz extracelular em Contextos individuais e multicelulares

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

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.

Medição simultânea em pH endocítica e citossólicos Compartimentos em células vivas usando microscopia confocal

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

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Medição dos Níveis de heme em células de mamíferos Síntese

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Medidas cálcio citosólico em células epiteliais renais por citometria de fluxo

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Análise de suspensões celulares isoladas de tecidos sólidos por citometria de fluxo espectral

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Imagens de alta resolução e Análise de individuais Astral microtúbulos Dynamics em brotamento Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

4D microscopia de levedura

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Efeito de proteínas fluorescentes na fusão parceiros usando Polyglutamine ensaios de toxicidade em levedura

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Medição das taxas de consumo de oxigênio em intacta Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...