Este trabalho foi financiado pelo NIH Grant 1R21AG031965-01A1. MK é um Ellison Medical Foundation Scholar novo no Envelhecimento.

Parte 1: Preparação das culturas de envelhecimento <ol><li> Cepas raia de interesse dos estoques congelados em placas de ágar YEPD (extrato de levedura 1%, 2% Bacto-peptona, agar 2%, 2% de glicose). </li><li> Incubar as células em 30 ˚ C durante 48 horas ou até colônias única aparecer. </li><li> Escolha colônias única e inocular em 5 mL de meio líquido YEPD (extrato de levedura 1%, 2% Bacto-peptona, 2% de glicose) em tubos de ensaio. </li><li> Crescer culturas durante a noite a 30 ˚ C, mantendo agitação constante utilizando um shaker ou tambor do rolo. </li><li> Inocular 5 mL de meio sintético (SC) completa (Tabela 1) com 50 mL da cultura durante a noite. Geralmente três culturas SC estão preparados para cada cepa para fornecer replicação triplicado biológica da análise de ciclo de vida de cada cepa sendo examinada. </li><li> Manter as culturas em 30 ˚ C com agitação constante em um monocilíndrico de todo o experimento (geralmente 2 ou mais semanas). </li></ol> Parte 2: Tomando um ponto de viabilidade idade Após dois dias de cultura em SC media, as células devem estar em fase estacionária e os primeiros anos de ponto está pronto para ser tomada. Subseqüentes idade pontos devem ser tomadas a cada 2-3 dias por um mínimo de duas semanas. Para cada ponto de idade: <ol><li> Prepare o Bioscreen 100 placas bem Honeycomb para inoculação preenchendo cada poço com 145 mL de YEPD. Certifique-se de deixar pelo menos um poço cheio de só YEPD e ausência de células para análise de dados mais tarde. </li><li> Remove as culturas de envelhecimento da incubadora. </li><li> Vortex brevemente a primeira cultura a ser inoculado na placa do favo de mel, tomando cuidado para não derramar da cultura. </li><li> Retire 5 mL da cultura mista e pipeta-lo no primeiro poço da placa do favo de mel. Chama boca de cada tubo de ensaio, antes e após a remoção da alíquota 5 mL. </li><li> Repita esse procedimento para cada cultura de envelhecimento sendo certo anotar a posição bem correspondentes a cada cultura. Idênticas posições bem deve ser usado para cada idade subseqüentes ponto durante todo o experimento. </li><li> Substituir as culturas para a 30 ˚ C incubadora quando terminar com a vacinação. </li></ol> Parte 3: Carregando o Bioscreen C máquina MBR <ol><li> Expor o compartimento incubadora, levantando a tampa e retire a tampa para a bandeja de amostra. </li><li> Inserir a placa recém-inoculados Honeycomb na bandeja de amostra (usar o slot da esquerda se você estiver apenas uma leitura da placa). </li><li> Recoloque a tampa para a bandeja de amostra e menor a tampa do compartimento de incubadora. </li><li> Verifique se o fluido de transferência de calor está acima do nível mínimo de preenchimento. Se baixo, adicionar mais usando uma pipeta de 1000 mL com o fluido de transferência de calor fornecido. </li><li> Usando o software Bioscreen &quot;EZExperiment&quot;, defina os parâmetros a seguir para obter as curvas de crescimento adequado para Saccharomyces cerevisiae: <ul><li> Número de amostras: Digite o número de poços com a mídia ou 200 </li><li> Filter: 420-580nm, Wideband </li><li> Temperatura: 30 ˚ C </li><li> Comprimento experimento: um dia, 0 horas, 0 segundos </li><li> Intervalo de medição: 30 minutos </li><li> Agitação: On, agitação contínua, de alta </li></ul></li><li> Clique em &quot;Iniciar&quot; para iniciar as leituras. </li></ol> Parte 4: Análise de Dados Tipicamente, seis idade pontos são tomadas ao longo de duas semanas. Os pontos de idade são tomadas nos dias 2, 4, 6, 9, 11 e 13. Dependendo do desenho experimental e tensões que está sendo testado, pode ser desejável para tomar idade pontos mais ou menos freqüentemente ou por mais de duas semanas. É importante carregar a placa do favo de mel na mesma ordem para todas as idades ponto tal que cada cultura envelhecimento corresponde à mesma posição em todas as idades bem-ponto, pois isso fará com que a análise de dados muito mais fácil. <ol><li> Obter os arquivos de saída da máquina Bioscreen MBR C. O software &quot;EZExperiment&quot; irá mostrar os dados Bioscreen como um arquivo delimitado por tabulação que é compatível com Microsoft Excel, assim como outros softwares. A primeira coluna mostra em que momento da medição OD foi tomada durante o experimento, com colunas subseqüentes representando cada poço nas placas Bioscreen Honeycomb (Figura 1). </li><li> Excluir a leitura OD primeiro de cada coluna. Esta leitura é &quot;ruído&quot;. </li><li> Normalizar os dados, subtraindo o valor da OD bem com YEPD sozinho de todos os valores de OD em cada coluna. Isso remove a absorvância de fundo pela mídia. </li><li> Plot as curvas conseqüência. As curvas mudará em função da idade (Figura 2). Por exemplo, traçando as curvas de crescimento da coluna 1 (poço 1 da placa do favo de mel) ao longo dos seis pontos do tempo, há uma nítida mudança para a direita das curvas ao longo do tempo. </li><li> Calcular o tempo de duplicação (δ) para cada poço com base na cinética de crescimento de dois dias a idade-ponto. A equação utilizada para calcular tempo de duplicação is: <img src="/files/ftp_upload/1156/equat.png" alt="Equação" /> onde OD 1 e 2 representam OD OD medições sucessivas e t 1 e t 2 é o tempo entre as medições. Calcule duplicação vezes apenas entre os valores OD de 0,2 para 0,5. A média desses valores é o tempo de duplicação para o bem. Calcule duplicação vezes apenas entre os valores OD de 0,2 para 0,5. A média desses valores é o tempo de duplicação para o bem. A maioria das estirpes do tipo selvagem levedura deve dar um valor de tempo de duplicação entre 85 a 90 minutos. </li><li> Para cada idade ponto, calcular a mudança de tempo (Dt) nas curvas de crescimento em relação à idade inicial de ponto (dia 2). Uma maneira fácil de fazer isso é determinar a diferença na duração do tempo que levou para cada poço para chegar a um OD de 0,3 entre os anos iniciais de ponto e cada subseqüente idade ponto. O tempo que um determinado poço atingiu uma OD de 0,3 pode ser calculado a partir da equação de regressão linear correspondente a ln (OD) em função do tempo entre os dois pontos temporais bracketing OD = 0,3. </li><li> Calcular a fração sobrevivente em cada idade ponto, a fim de gerar uma curva de sobrevivência (Figura 3). Definir o ponto inicial idade (dia 2) a ser viabilidade de 100%. Para cada idade sucessiva ponto-calcular a sobrevivência por cento usando a equação: <img src="/files/ftp_upload/1156/equat2.png" alt="Equação segunda" width="245" height="115" /> s onde n é a porcentagem de sobrevivência, Dt n é a mudança da hora, e n δ é o tempo de duplicação. </li><li> Gerar curvas de sobrevida (Figura 3B), como desejado para cada bem (ou poços replicar) traçando a fração (ou percentual) de células viáveis ​​em função da idade. </li><li> Calcular a sobrevivência integral (SI) para cada poço. SI é definida como a área sob a curva de sobrevivência e pode ser estimado pela fórmula: <img src="/files/ftp_upload/1156/equat3.png" alt="Equação 3" width="400" height="79" /> onde a idade n é a idade-ponto (eg 2, 4, 6, 9, 11 e 13) e s n é o valor de sobrevivência nessa idade ponto. </li><li> Determinar os parâmetros estatísticos de poços replicar a partir dos dados do SI e sobrevivência. Comum parâmetros estatísticos de interesse incluem mediana, média e variância para cada conjunto de fatores biológicos repetições. A t-teste ou análise similar pode ser usado para comparação de pares de SI para fins experimentais e grupos controle. Também pode ser desejável para gerar curvas de sobrevivência, como descrito em 4.8 de média biológica repetições. </li></ol> Parte 5: Os resultados representativos Ao término do experimento, você terá traçado da curva de sobrevivência e realizada a análise de dados suficientes para determinar o potencial de envelhecimento cronológico de várias estirpes diferentes ou condições. Se realizada corretamente, as curvas de crescimento obtidas a partir da máquina Bioscreen MBR C deve ser semelhante ao mostrado na Figura 2 e as curvas de sobrevivência resultante deve ser semelhante aos mostrados na Figura 3. Geralmente, do tipo selvagem células cultivadas nas condições descritas aqui terão uma duração de vida média da ordem cronológica de 7 dias. Variação substancial na sobrevida é observado em cepas diferentes e sob algumas condições, tais como o crescimento em meios de glicose 0,05%, a sobrevida mediana pode exceder 30 dias. <img src="/files/ftp_upload/1156/1156fig1.png" alt="" /> Figura 1. Saída de dados do Bioscreen programa &quot;EZExperiment&quot;. A coluna mostra o momento em que uma leitura de absorbância foi tirada. Sucessivas colunas representam os poços da placa do favo de mel inoculados com células retiradas de culturas de envelhecimento. <img src="/files/ftp_upload/1156/1156fig2.png" alt="" /> Figura 2. Curvas de crescimento a partir de um único replicar biológica ao longo de uma experiência. Há uma clara mudança nas curvas ao longo do tempo como as células do envelhecimento na cultura perdem a viabilidade. O período de tempo entre o ponto de tempo inicial (dia 2) e um ponto de tempo sucessivos determina viabilidade nessa idade particular. <img src="/files/ftp_upload/1156/1156fig3a.png" alt="" /><img src="/files/ftp_upload/1156/1156fig3b.png" alt="" /> Figura 3. A) A curva de sobrevida usando os dados gerados conseqüência da Figura 1. No dia tempo 2 é definido como o ponto de viabilidade de 100%. B) curvas de sobrevivência final de duas cepas testadas no mesmo experimento. Essas curvas representam as viabilidades de sobrevivência média de três repetições para cada biológica tensão. Barras de erro representam o desvio padrão dentro biológica repetições. A área sombreada sob a curva de sobrevivência representa a sobrevivência integral (SI) para a cepa 1. <table border="1"><tbody><tr><td colspan="2"> Tabela 1. Médio Definido sintético usado para estudos de envelhecimento cronológico (cepa background BY4743) </td></tr><tr><td> Componente </td><td> Concentração (g / L) </td></tr><tr><td> D-glucose </td><td> 20 </td></tr><tr><td> Base de levedura de nitrogênio (-AA/AS) </td><td> 1,7 </td></tr><tr><td> (NH 4) 2 SO 4 </td><td> 5,0 </td></tr><tr><td> Adenina </td><td> 0,04 </td></tr><tr><td> L-Arginina </td><td> 0,02 </td></tr><tr><td> Ácido L-aspártico </td><td> 0,1 </td></tr><tr><td> Ácido L-glutâmico </td><td> 0,1 </td></tr><tr><td> L-Histidina </td><td> 0,1 </td></tr><tr><td> L-leucina </td><td> 0,3 </td></tr><tr><td> L-Lisina </td><td> 0,03 </td></tr><tr><td> L-Metionina </td><td> 0,02 </td></tr><tr><td> L-Fenilalanina </td><td> 0,05 </td></tr><tr><td> L-Serina </td><td> 0,375 </td></tr><tr><td> L-Treonina </td><td> 0,2 </td></tr><tr><td> L-Triptofano </td><td> 0,04 </td></tr><tr><td> L-Tirosina </td><td> 0,03 </td></tr><tr><td> L-valina </td><td> 0,15 </td></tr><tr><td> Uracil </td><td> 0,1 </td></tr></tbody></table> Nota: Esta receita é responsável por auxotrophies em linhagem diplóide BY4743. Auxotrophies aminoácido deve ser compensado pela adição de uma concentração 5X final para o meio sintético completo.

<ol>
	<li>Steinkraus, K. A., Kaeberlein, M., Kennedy, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicative+aging+in+yeast:+the+means+to+the+end.">Replicative aging in yeast: the means to the end.</a> Annu Rev Cell Dev Biol. 24, 29 (2008).</li><li>Kaeberlein, M., Conn, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of models for human aging. , 109 (2006).</li><li>Fabrizio, P., Longo, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chronological+life+span+of+Saccharomyces+cerevisiae.">The chronological life span of Saccharomyces cerevisiae.</a> Aging Cell. 2 (2), 73 (2003).</li><li>Murakami, C. J., Burtner, C. R., Kennedy, B. K., Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+high-throughput+quantitative+analysis+of+yeast+chronological+life+span.">A method for high-throughput quantitative analysis of yeast chronological life span.</a> J Gerontol A Biol Sci Med Sci. 63 (2), 113 (2008).</li><li>Piper, P. W., Harris, N. L., MacLean, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preadaptation+to+efficient+respiratory+maintenance+is+essential+both+for+maximal+longevity+and+the+retention+of+replicative+potential+in+chronologically+ageing+yeast.">Preadaptation to efficient respiratory maintenance is essential both for maximal longevity and the retention of replicative potential in chronologically ageing yeast.</a> Mech Ageing Dev. 127 (9), 733 (2006).</li><li>Fabrizio, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+is+a+mediator+of+an+altruistic+aging+program+in+Saccharomyces+cerevisiae.">Superoxide is a mediator of an altruistic aging program in Saccharomyces cerevisiae.</a> J Cell Biol. 166 (7), 1055 (2004).</li></ol>

O alto rendimento cronológica ensaio expectativa de vida aqui descrito é um método eficaz para quantificar o potencial de envelhecimento de um grande número de cepas com alta precisão e de precisão. O principal avanço deste método em relação aos métodos clássicos de determinação de sobrevivência através da contagem de unidades formadoras de colônia (por exemplo, ver 3) é o uso de um dispositivo de leitura shaker / incubadora / placa, como a máquina Bioscreen MBR C para obtenção de curvas ...

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<td>Bacto Peptone</td>
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quantifying yeast chronological life span by outgrowth of aged cells

A levedura de brotamento<em&gt; Saccharomyces cerevisiae</em&gt; Provou ser um organismo modelo importante no campo da pesquisa sobre o envelhecimento<sup&gt; 1</sup&gt;. O replicativa e expectativa de vida cronológica são dois paradigmas estabelecidos usado para estudar o envelhecimento em leveduras. Envelhecimento replicativo é definido como o número de células filhas uma célula mãe solteira fermento produz antes de senescência; envelhecimento cronológico é definido pelo comprimento das células de tempo podem sobreviver em uma não-divisão, o estado de quiescência-like<sup&gt; 2</sup&gt;. Nós desenvolvemos um método de alto rendimento para a determinação quantitativa do tempo de vida cronológica. Este método envolve o envelhecimento das células em um meio definido sob agitação e à temperatura constante. Em cada idade-ponto, uma sub-população de células é retirado da cultura de envelhecimento e inoculadas em meio de cultura rico. A alta resolução curva de crescimento é, então, obtido para esta sub-população de células envelhecidas usando uma máquina Bioscreen C MBR. Um algoritmo é então aplicado para determinar a proporção relativa de células viáveis ​​em cada sub-população com base na cinética de crescimento em cada idade-ponto. Este método requer substancialmente menos tempo e recursos em comparação com outros ensaios cronológica vida útil, mantendo a reprodutibilidade e precisão. A natureza de alto rendimento deste ensaio devem permitir grande escala telas genética e química para identificar modificadores de longevidade da novela para mais testes em organismos mais complexos.

Watch this Scientific Journal Video about Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells at JoVE.com

Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

Envelhecimento cronológico em levedura refere-se à perda de viabilidade celular associada ao tempo na fase estacionária. Aqui nós descrevemos um método de alto rendimento para determinar quantitativamente fermento esperança de vida cronológica.

Quantificar Yeast Life Span cronológica por conseqüência de células envelhecidas

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and ...

quantifying-yeast-chronological-life-span-by-outgrowth-of-aged-cells

Research

JoVE Journal

Biology

16.5K visualizações.  University of Washington. Envelhecimento cronológico em levedura refere-se à perda de viabilidade celular associada ao tempo na fase estacionária. Aqui nós descrevemos um método de alto rendimento para determinar quantitativamente fermento esperança de vida cronológica.

Vídeo: Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

Uma técnica rápida para a visualização de células vivas de levedura Imobilizado

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

Biologia

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Medição Caenorhabditis elegans Expectativa de Vida em meios sólidos

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

Medição Span vida replicativa na levedura de brotamento

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Purificação de mitocôndrias de células de levedura

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Medição Caenorhabditis elegans Expectativa de Vida em 96 placas de microtitulação Bem

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

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 ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

A quantificação temporal da expressão MAPK induzida em células de levedura Solteiro

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

Geração e cultura de sangue Conseqüência células endoteliais do sangue periférico humano

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.