We would like to thank the Children&#8217;s Hospital at Montefiore, Department of Pediatrics, for providing support for these studies. We would like to thank the National Alopecia Areata Foundation for providing financial support for conducting studies with C3H/HeJ engrafted mice.

Declaração de Ética: Todos os estudos que envolvam animais devem ser aprovados pelo IACUC da instituição (para os dados que se segue, os protocolos foram aprovados pela Montefiore IACUC, protocolo nº 11-6-240 e # 13-7-100). Animais são fornecidos anestesia luz, como indicado para o único propósito de mantê-los ainda durante a fotografia, não há procedimentos dolorosos necessários para este protocolo. 1. Aquisição de fotografias <ol><li> Definir o foco e campo de visão para imager gel usando papel com texto impresso. Verifique uniformidade da fonte de luz em toda a região fotografada. Para ajudar a garantir a uniformidade, usar um gerador de imagens gel com um construído em fonte de luz para a fotografia reflexiva. Não utilize uma fonte de luz transillumination, pois isso criaria uma silhueta do animal que não é adequado para uma análise mais aprofundada em tons de cinza. NOTA: Isto irá colocar o mouse ligeiramente fora de foco, que fornece a média óptica em toda a região de interesse (ROI) e vontadeajudar a reduzir erros quantais para regiões muito pequenas (isto é, &lt;10 pixels) de interesse. Regiões maiores de interesse não será afetado por este menor focando ajuste, nem serão afetados por diferenças no tamanho do animal. </li><li> Anestesiar os animais utilizando cetamina (100 mg / mL) / xilazina (20 mg / mL) (2: 1), pois isso proporciona um efeito de anestesia e uma recuperação rápida rápida e é ideal para fotografar vários animais. NOTA: A anestesia é confirmada quando o animal ainda é suficiente para permitir a fotografia. Como os animais tipicamente recuperar desta anestesia luz dentro de 10-15 min, o veterinário não recomenda o uso de pomadas para os olhos. </li><li> Coloque animais anestesiados em imager gel em alinhamento vertical (o mais próximo possível paralelo quanto possível). <ol><li> Para fotografias dorsais, colocar os animais em decúbito ventral, com os membros estendidos. </li><li> Para fotografias ventral, colocar os animais em decúbito dorsal, tomando cuidado para que os animais não são rodado lateralmente. </li&gt; </ol></li><li> Coloque padrão em tons de cinza na região fotografada. </li><li> Feche a porta de acesso. Importante: A luz ambiente pode apresentar variações na exposição. </li><li> Defina F-stop para uma exposição que coloca a região de interesse dentro da faixa linear de aquisição (leitura F-stop). NOTA: A maioria dos sistemas irá mostrar onde a imagem está saturada. </li><li> Tirar a fotografia. </li><li> Alterar F-stop para outra configuração de exposição que coloca a região de interesse dentro do intervalo linear, aumentando ou diminuindo o F-stop por 1, tal que tanto o padrão e a região de interesse permanece num intervalo linear de aquisição (referência F- pare). </li><li> Tirar a fotografia. </li><li> Uma vez que a fotografia é concluída, colocar os animais em uma tabela de aquecimento e monitorar até que possam manter decúbito esternal. Retorno animais para suas gaiolas. Retorne o grupo de animais ao biotério uma vez que todos os animais são totalmente recuperado. </li></ol> 2. Quantificação da absorção de luz &lt;/ P&gt; <ol><li> Marcar regiões de interesse nas imagens dos animais utilizando software fornecido para imager gel. <ol><li> Para ver o animal inteiro dorsal, utilizar uma imagem rectangular ou oval que se estende a partir das extremidades superiores dos membros inferiores, que se prolonga lateralmente, tanto quanto possível, de tal modo que nenhuma parte da caixa se estende para além da parte de trás do animal, tal como mostrado na Figura 1A. NOTA: Pode-se também marcar a área de interesse, utilizando uma ferramenta de desenho à mão livre. </li><li> Para animal inteiro ventral vista, use 2 retângulos: uma que cobre a região pélvica e um cobrindo a área do peito, como mostrado na Figura 1B. </li><li> Para uma região menor de interesse, ou seja, o local da administração da droga, marca, conforme apropriado. </li></ol></li><li> Região (s) Mark de juros sobre o padrão de absorção em tons de cinza. </li><li> Absorção da ficha de cada uma das regiões marcadas de interesse. </li></ol> 3. Análise Níveis de absorção obtained para as regiões de interesse podem necessitar de ser normalizadas para o padrão de fundo para comparação entre as fotografias. A relação de absorção é a exposição log-log (isto é, a relação é linear entre o log (exposição) e log (absorção)). Usando esta relação, a absorção da região de interesse pode ser normalizada a um padrão de valor de fundo, o que permitirá que as absorções de ser directamente comparados entre diferentes, incluindo as fotografias tomadas em diferentes pontos de tempo (isto é, medidas em série dentro de um protocolo de estudo). O procedimento para a execução dessas correções é detalhada em etapas opcionais 3.1 e 3.2. <ol><li> (Opcional) curva Lote de log (exposição) vs. log (absorção), utilizando os valores obtidos a partir de padrão de absorção em tons de cinza. </li><li> (Opcional) Ajuste para variações no padrão de cada fotografia da seguinte forma: <ol><li> Selecione F-stop para a leitura (como determinado na etapa 1.6) e F-stop para a referência (conforme determinado no step 1,8). </li><li> Calcule a absorção média do padrão em todas as fotografias na leitura de F-stop. Esta média é o Valor de Referência Padrão (RSV). </li><li> Calcular diferença de absorção entre a leitura e de referência configurações F-stop em cada fotografia para o padrão (delta-S) e para cada ROI definido (delta-ROI-1, delta-ROI-2 ... ..delta-ROI-X) </li><li> Calcule a absorção corrigido para cada ROI da seguinte forma: de absorção corrigido (ROI-X) = Absorção (ROI-X) com a leitura F-stop + (RSV - absorção de padrão na leitura de F-stop) * (delta-ROI-X / delta-S) </li></ol></li><li> Compilar os dados experimentais e executar análise de dados usando técnicas estatísticas padrão para variáveis ​​contínuas (ie, ANOVA, T-teste). </li></ol>

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Authors have nothing to disclose.

Neste relatório, uma descrição detalhada é fornecida de uma nova técnica para quantificar a perda de cabelo em roedores. Esta técnica utiliza um gerador de imagens de gel para aquisição e análise de imagem, o equipamento que está prontamente disponível, na maioria dos laboratórios. As medições foram mostrados para ser robusto para pequenas variações na técnica (Figuras 4-5), e está bem correlacionada com o grau de perda de cabelo visual em modelos de tratamento (Figura 6).</str...

Alopecia (queda de cabelo) pode ser um evento psicologicamente e emocionalmente estressante com múltiplas causas. Calvície de padrão masculino é a causa mais comum de alopecia, que afecta cerca de dois terços do sexo masculino de 35 anos 1. Um padrão semelhante de perda de cabelo pode ser observado em mulheres com síndrome do ovário policístico. Em ambos estes distúrbios, a perda de cabelo é mediada androgénio. Alopecia também pode ocorrer como uma doença auto-imune, alopecia areata, que afecta 1,7% da população 2. Alopecia pode ocorrer como um efeito colateral de alguns tratamentos médicos, tais como a quimioterapia e 3. Uma elevada percentagem (65-85%) dos pacientes de quimioterapia experimentam algum grau de alopecia 4,5. As consequências psicológicas da perda de cabelo foram bem estudados no cenário quimioterapia. Alopecia induzida pela quimioterapia pode resultar em ansiedade, depressão, uma imagem corporal negativa, baixa auto-estima e um senso reduzido de bem-estar 6,7. Uma alta eprcentage (47-58%) de pacientes com câncer feminino considerar a perda de cabelo para ser o aspecto mais traumático da quimioterapia, e até 8% declínio tratamento por medo de perda de cabelo 4,6. Há também evidências de alopecia androgênica para apoiar a terapia para reduzir as conseqüências psicológicas e até mesmo médicos de perda de cabelo 8,9. Da mesma forma, a alopecia areata foi reportado ter consequências psicológicas graves 2, e a natureza irregular da perda de cabelo pode levar a um resultado estético mais desagradável do que a maioria das outras causas de perda de cabelo. Enquanto as drogas com efeitos anti-androgênicos leves (ie, espironolactona) tinham sido utilizados com sucesso limitado como terapia para alopecia, o primeiro medicamento eficaz para alopecia foi minoxidil 10. Este anti-hipertensora tem um efeito secundário observado de provocar o crescimento do cabelo, e é agora utilizada como terapia tópica para muitas formas de alopecia. No entanto, as respostas são muitas vezes incompleta, com alguns assuntos mostrando apenas lentoção de perda de cabelo em vez de rebrota real 10. Finasteride é um antagonista competitivo de tipo II 5α-redutase, que bloqueia a conversão de testosterona em di-hidrotestosterona, resultando em melhorias na alopecia androgénica, em detrimento de androgénio bloqueio parcial sistémica. As taxas de resposta com a longo prazo (10 anos), a terapia são em torno de 50% 11. No geral, apesar de considerável pesquisa nesta área, ainda não há uma terapia adequada para a perda de cabelo. Durante décadas, cientistas e médicos examinaram métodos de medir o crescimento do cabelo do couro cabeludo em ensaios clínicos. Com o desenvolvimento de drogas que tratam alopecia, tem havido uma necessidade maior de meios fiáveis, económicos e minimamente invasivas de medição do crescimento do cabelo e, especificamente, a resposta à terapia. Tecnologia de análise de imagem para uma quantificação precisa de densidade do cabelo em pacientes com distúrbios de perda de cabelo produziu resultados consistentes e válidos no passado usando uma variedade de techniques, incluindo a análise de imagens digitalizadas 12, análise de imagem de cabelos individuais e lesões cutâneas 13, e digitalização microscópica para quantificar a massa de cabelo em uma região do couro cabeludo definido 14. Infelizmente, enquanto as metodologias acima têm proporcionado uma melhor avaliação da eficácia para o cabelo crescimento promovendo intervenções em ensaios clínicos, estes métodos não tenham sido aplicadas a estudos com roedores em investigações pré-clínicas. Nosso objetivo é desenvolver um método consistente e confiável para quantificar a perda de cabelo em ratos, o que permitirá que os investigadores a avaliar com mais precisão e comparar novas abordagens terapêuticas para várias formas de alopecia. Nós desenvolvemos uma metodologia utilizando equipamento prontamente disponível na maioria dos laboratórios que permitam a quantificação rápida e confiável de densidade de cabelo em ratos com cabelo marrom ou preto. Esta metodologia foi testada em modelos de rato de alopecia induzida por quimioterapia, alopécia areata e alopécia de waxing. Um protocolo detalhado é apresentado para a execução dessas medidas, incluindo dados de validação de C57BL / 6 e C3H / HeJ cepas de camundongos. Como esta técnica baseia-se na detecção de absorção de luz a partir de pigmentos na haste do cabelo, que não pode ser usado para detectar o crescimento de cabelo em ratinhos ou ratos albinos brancos.

<table border="0" cellpadding="0" cellspacing="0" width="623">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:121px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">KODAK Gel Logic 100 Imaging System&#160;&#160;</td>
			<td style="width:121px;">Eastman Kodak Company, Rochester, NY, USA</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Gel imager for obtaining and analyzing photographs, must have built in light source for reflective photography.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">The Kodak/Tiffen Q-13 Gray Scale</td>
			<td style="width:121px;">Imatest, http://www.imatest.com/docs/q13/</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Greyscale standard</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">C57BL/6J Mice</td>
			<td style="width:121px;">&#160;Jackson Laboratories, Bar Harbor, Maine</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Mice for representative study</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">C3H/HeJ engrafted mouse</td>
			<td style="width:121px;">&#160;Jackson Laboratories, Bar Harbor, Maine</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Mice for representative study</td>
		</tr>
	</tbody>
</table>

a new technique for quantitative analysis of hair loss in mice using grayscale analysis

Alopecia é uma forma comum de perda de cabelo, que pode ocorrer em diversas condições, incluindo a perda de padrão masculino de cabelo, síndrome do ovário policístico, e alopecia areata. Alopecia também pode ocorrer como um efeito secundário da quimioterapia em pacientes com cancro. Neste estudo, o nosso objectivo foi desenvolver um método consistente e fiável para quantificar a perda de cabelo em ratinhos, o que vai permitir aos investigadores comparar e avaliar com precisão a novas abordagens terapêuticas para estas várias formas de alopécia. O método utiliza um gerador de imagens de gel padrão de obtenção e processamento de imagens de ratos, medindo a absorção de luz, o que ocorre em proporção com a quantidade aproximada de cabelo preto (ou cinzento) no rato. Os dados que foram quantificados desta forma podem ser analisados ​​utilizando técnicas estatísticas padrão (ou seja, ANOVA, teste t). Esta metodologia foi testada em modelos de rato de alopecia induzida por quimioterapia, alopécia areata e alopécia de depilação. Neste relatório, o protocolo detalhado é prestura orientada para a execução dessas medidas, incluindo dados de validação de C57BL / 6 e C3H / HeJ cepas de camundongos. Esta nova técnica oferece uma série de vantagens, incluindo a relativa simplicidade de aplicação, a confiança no equipamento que está prontamente disponível na maioria dos laboratórios de pesquisa, e aplicar uma avaliação objectiva, quantitativa que é mais robusto do que avaliações subjetivas. Melhorias na quantificação do crescimento do cabelo em camundongos vai melhorar estudo de modelos de alopecia e facilitar a avaliação de novas terapias promissoras em estudos pré-clínicos.

Esta técnica foi validado utilizando camundongos C3H / HeJ em vós implantada, a modelo de rato com alopecia areata 16. Estes animais desenvolvem a perda de cabelo global que progride de forma gradual ao longo do tempo. No entanto, a perda de cabelo ocorre em manchas, e pode variar de um rato para o outro, a introdução de uma grande variabilidade e dificulta as avaliações qualitativas. Este modelo proporcionou uma oportunidade para testar as correlações entre as medições de densidade óptica em difer...

Watch this Scientific Journal Video about A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis at JoVE.com

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Uma nova técnica de Análise Quantitativa de perda de cabelo em ratos usando Análise Tons de Cinza

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

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8.9K visualizações.  Children's Hospital at Montefiore. Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Vídeo: A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A Neuronal e astrocitária Ensaio Co-Cultura de Análise de Conteúdo de Alta de neurotoxicidade

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biologia

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Em células vivas, Imagem e Análise Quantitativa de células embrionárias epiteliais em Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Quantitativas de células vivas de Análise de microscopia de fluorescência de levedura

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Análise Quantitativa de Migração aleatória de células usando microscopia de lapso de tempo vídeo

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

Um fluxo de trabalho Análise Quantitativa de Fitness

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Análise Genética rápida de epitelial-mesenquimal sinalização durante a regeneração do cabelo

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Análise Quantitativa de Autophagy usando microscopia de fluorescência avançada 3D

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualização e análise de moléculas de mRNA Usando Fluorescência<em&gt; In Situ</em&gt; A hibridação em<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

Uma Nova Abordagem para a Análise Comparativa de Complexos Multiproteicos Baseado em<sup&gt; 15</sup&gt; N Metabólica Labeling e quantitativa Espectrometria de Massa

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Um Ensaio de Ligação de Alto Rendimento II do MHC para análise quantitativa de Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Uma nova técnica de Análise Quantitativa de perda de cabelo em ratos usando Análise Tons de Cinza

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