O trabalho no laboratório dos autores foi apoiado pela Deutsche Forschungsgemeinschaft (http://www.dfg.de; CO 1139/1-1), a Fonds der Chemischen Industrie (http://www.vci.de/fonds) e o Centro de Biologia Molecular de Göttingen (GZMB). Os autores gostariam de reconhecer Jörg Stülke por comentários úteis e leitura crítica do manuscrito.

1. Preparação de Placas de Ágar, Mídia de Cultura, Criostocks e Pré-Culturas
<ol><li>Prepare a mídia de crescimento e os reagentes necessários (ver tabela de materiais e reagentes).</li>
	<li>Listrar as cepas B. subtilis (por exemplo. BP40 (rocG+ gudBCR amyE::PgudB-yfp) e BP52 (rocG+ gudB+ ame::PgudB-cfp) expressando um e dois GDHs ativos,respectivamente) 2 que serão usados no experimento de competição em placas de ágar médio sp para obter colônias únicas. Incubar as placas durante a noite a 37 °C.</li>
	<li>Pegue colônias simples e tubos de cultura estéril inoculados contendo meio líquido de 4 ml LB. Cresça as bactérias durante a noite a 28 °C e 220 rpm.</li>
	<li>Cresça culturas durante a noite a 28 °C para evitar que as células lise ou esporulam.</li>
	<li>Meça a densidade óptica das culturas em um comprimento de onda de 600 nm (OD600) usando um espectrofotômetro padrão.</li>
	<li>Se as culturas atingiram um OD600 de 2.0, misture 0,75 ml das culturas com 0,75 ml de uma solução estéril de 50% de glicerol em tubos de reação de 1,5 ml. O OD600 final deve ser 1.0 para obter criostocks contendo cerca de 108 células/ml.</li>
	<li>Armazene os tubos a -80 °C.</li>
	<li>Faça três pré-culturas de cada cepa rotulada com os genes de fluoróforo de codificação cfp e yfp. Inocular as pré-culturas (tubos de cultura estéril contendo meio líquido de 4 ml LB) com células de 1 μl a partir de -80 °C criostocks. Incubar as culturas durante a noite a 28 °C e 220 rpm.</li>
</ol>2. Cocultivação de Bactérias, Coleta de Amostras e Armazenamento de Amostras
<ol><li>Prepare recentemente 100 ml C-Glc e CE-Glc meio mínimo (ver tabela de reagentes e materiais), e transfira 20 ml de cada meio em frascos de shake estéreis de 100 ml.</li>
	<li>Pegue 0,1 ml das préculturas que foram cultivadas durante a noite, dilui-as com 0,9 ml de lb médio em um cuvette de 1,5 ml, e determine o OD600.</li>
	<li>Para o experimento de competição, pegue essas pré-culturas das diferentes cepas que têm um OD600 semelhante entre 1,0-1,5.</li>
	<li>Para obter populações de células mistas, diluir as células das préculturas que tinham o OD600 adequado para um OD600 de 0,05 em 20 ml C-Glc e CE-GLC meio mínimo suplementado em frascos de shake de 100 ml. Para o experimento de competição, as duas cepas devem ser misturadas em uma proporção de 1:1.</li>
	<li>Pegue 10 ml de amostras dos frascos, transfira-as para tubos plásticos de 15 ml, colmeia as células por centrifugação por centrifugação por 10 minutos a 4.000 x g em uma centrífuga superior de mesa padrão e descarte o supernascedor.</li>
	<li>Resuspenque as células em 0,5 ml de meio LB fresco, e transfira as células para um copo de reação estéril de 1,5 ml. Adicione 0,5 ml de glicerol 50% estéril, misture a suspensão por um rigoroso vórtice e armazene as amostras em um congelador de -80 °C até um novo tratamento.</li>
	<li>Incubar as células que são deixadas nos frascos de shake por até 24 horas a 37 °C e 220 rpm. Mantenha os frascos de agitação no escuro para evitar o branqueamento fotográfico dos fluoroforos.</li>
	<li>Pegue mais amostras de 0,1 ml após 7 horas e 24 horas de crescimento. Meça e observe o OD600 de diluições de 1:10 (em C-Glc ou CE-Glc médio mínimo) das amostras.</li>
	<li>Pegue a quantidade apropriada de células de cada cultura para fazer criostocks que têm um OD600 de 1.0. Armazene as amostras a -80 °C até um novo tratamento.</li>
</ol>3. Tratamento amostral, chapeamento e incubação para análises quantitativas
<ol><li>Depois de coletar todas as amostras, descongele os criostocks e dilua as células em uma solução salina de 0,9% (ver tabela de reagentes e materiais) até 10-3.</li>
	<li>Placa 0,1 ml das diluições de 10-3 em placas de ágar médio sp e distribuir as células usando pipetas de vidro estéreis.</li>
	<li>Incubar as placas durante a noite a 37 °C no escuro até que colônias únicas tenham aparecido.</li>
</ol>4. Contando os Sobreviventes por Microscopia de Fluorescência Estéreo para Análise Quantitativa
<ol><li>Divida a parte inferior da placa de ágar com uma caneta preta em quatro partes para uma melhor orientação enquanto conta os sobreviventes sob o microscópio de fluorescência estéreo.</li>
	<li>Coloque a placa de cabeça para baixo sob o microscópio e coloque as colônias em foco usando a fonte de luz fria.</li>
	<li>Uma vez que as colônias estejam em foco, mude para o conjunto de filtros apropriado para CFP para visualizar as células sobreviventes da cepa rotulada cfp. Conte os sobreviventes desta cepa rotulando as colônias com uma caneta e note o número.</li>
	<li>Remova as etiquetas com etanol e mude para o conjunto de filtros YFP para visualizar as células sobreviventes da cepa rotulada por yfp. Conte os sobreviventes novamente rotulando as colônias com uma caneta e note o número.</li>
</ol>5. Tratamento amostral e Microscopia para Análises Semiquantitativas
<ol><li>Para figuras ilustrativas, local 10 μl da diluição de 10-4 (aproximadamente 100 células) do passo 3.1 em uma placa de ágar SP (ver Figura 3).</li>
	<li>Incubar as placas durante a noite a 37 °C no escuro até que colônias únicas tenham aparecido.</li>
	<li>Coloque a placa de Petri sem tampa sob o microscópio e coloque o local em foco usando a fonte de luz fria.</li>
	<li>Tire fotos dos pontos para figuras ilustrativas. Escolha um tempo de exposição adequado para tirar as fotos das colônias com a fonte de luz fria.</li>
	<li>Sem mover a placa, mude para o conjunto do filtro CFP, ajuste o tempo de exposição e tire uma foto. Faça o mesmo com o conjunto de filtros YFP e guarde as imagens para análises posteriores.</li>
</ol>6. Análise de dados
<ol><li>Use softwares como o Excel para análises quantitativas de dados. Com base nas colônias contadas, calcule a porcentagem de colônias amarelas e azuis em relação ao número total de colônias, que estão fixadas em 100%.</li>
	<li>Use os números calculados para criar um diagrama de barra empilhado (ver Figura 4 e Figura 5). Use um programa de processamento de imagens como o Adobe Photoshop para construir fotos mescladas das fotos tiradas dos diferentes pontos. Alternativamente, o software ImageJ disponível livremente, pode ser usado <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> para processamento de imagens.</li>
	<li>Abra as imagens de um ponto que foram tiradas com o conjunto de filtros CFP e o conjunto de filtros YFP. Otimize o contraste e o brilho para reduzir qualquer fluorescência de fundo da mídia.</li>
	<li>Vá a uma das fotos e selecione todas. Copie a foto e cole-a na outra foto.</li>
	<li>Use a função "esquiva de cor" para mesclar as imagens ou sobrepor as imagens fluorescentes usando a guia canais. Imagens mescladas das colônias de diferentes mídias e diferentes pontos de tempo representam o crescimento das diferentes cepas dentro da cultura líquida.</li>
</ol>7. Dicas específicas: Experimento do interruptor de corante e cocultivação de cepas isogênicas rotuladas com cfp e yfp
A expressão de qualquer um dos dois genes codificadores de fluoróforos em B. subtilis pode influenciar o condicionamento físico e, portanto, a taxa de crescimento das bactérias. Portanto, recomenda-se realizar os seguintes experimentos, a fim de excluir que a eliminação de uma cepa concorrente da população celular durante o cultivo é simplesmente devido a um efeito negativo do fluorohore:
<ol><li>Repita todo o experimento e cocultivamente rotulado cepas (por exemplo, a cepa anteriormente rotulada cfpé agora rotulada com o gene yfp e vice-versa). Embora inversos, os resultados obtidos devem ser comparáveis à observação anterior de que uma cepa tem uma vantagem de crescimento seletiva sobre a outra cepa.</li>
	<li>Repita todo o experimento e cocultivato cepas isogênicas rotuladas com yfp e cfp (por exemplo, BP40 (rocG+ gudBCR amyE::PgudB-yfp) e BP41(rocG+ gudBCR amyE::PgudB-cfp)). Estimar o efeito negativo de qualquer um dos dois fluoroforos na aptidão das bactérias seguindo a composição da população celular ao longo do tempo.</li>
</ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Vários métodos foram desenvolvidos para analisar a aptidão competitiva das bactérias16. Em muitos casos, as bactérias foram rotuladas com diferentes fitas de resistência a antibióticos17. Semelhante à nossa abordagem, a rotulagem das células com fitas de resistência a antibióticos permite a avaliação da aptidão competitiva das bactérias durante a cocultivação em condições de crescimento definidas. Além disso, este método pode ser usado para determinar a aptidão competitiva das ...

As bactérias do solo são geralmente dotadas de redes regulatórias flexíveis e amplas capacidades metabólicas. Ambas as características permitem que as células ajustem suas vias catabólicas e anabólicas para competir com seus companheiros e outros microrganismos pelos nutrientes, que estão disponíveis em um determinado nicho ecológico1. No entanto, se as bactérias não conseguem se adaptar ao seu ambiente, outros mecanismos podem explicar a sobrevivência de uma espécie. De fato, como muitas bactérias se proliferam rapidamente e as populações podem atingir altas densidades celulares, subpopulações podem ter mutações benéficas acumuladas espontaneamente que fornecem às células uma vantagem de crescimento seletiva e, portanto, aumentam sua aptidão. Além disso, hotspots mutacionais e mutagênese adaptativa induzida pelo estresse podem facilitar a evolução de uma bactéria mal adaptada2,3. Assim, o acúmulo de mutações e crescimento sob seleção contínua é a origem da enorme diversidade microbiana, mesmo dentro do mesmo gênero4,5. Como na natureza, a modelagem de genomas bacterianos também ocorre em laboratório devido ao cultivo contínuo em seleção. Isso é exemplificado pela domesticação da bactéria Gram-positive B. subtilis, que é usada mundialmente em pesquisa básica e na indústria. Na década de 1940, a subtilis foi tratada com raios-X prejudiciais ao DNA, seguido pelo cultivo sob uma condição específica de crescimento6. As mutações que se acumularam nas bactérias durante sua domesticação são responsáveis pela perda de muitas características de crescimento, ou seja, a cepa de laboratório B. subtilis 168 perdeu a capacidade de formar colônias complexas7,8.
Hoje em dia, para as bactérias modelo mais bem estudadas Escherichia coli e B. subtilis,uma variedade de ferramentas poderosas está disponível para manipular geneticamente seus genomas a fim de abordar questões científicas específicas. Às vezes, a inativação de um gene de interesse causa um grave defeito de crescimento, que é então claramente visível no meio de crescimento padrão9. Em contraste, mutações que causam um fraco defeito de crescimento e, portanto, apenas afetam ligeiramente o condicionamento físico da cepa são muitas vezes ignoradas. No entanto, em ambos os casos a incubação prolongada e a passagem das cepas mutantes por várias gerações geralmente resultam no acúmulo de mutantes supressores que restauraram o fenótipo da cepa dos pais2,9. A caracterização dos mutantes supressores e a identificação das mutações que restauraram o defeito de crescimento da cepa mutante dos pais é uma abordagem muito útil que permite a elucidação de processos celulares importantes e muitas vezes novos10,11.
Estamos interessados no controle da homeostase glutamato em B. subtilis12. Semelhante a E. coli, B. subtilis responde à perturbação da homeostase glutamato (ou seja,bloco na degradação do glutamato2) pelo acúmulo de mutantes supressores. As alterações genômicas nesses mutantes supressores que foram adquiridos por mutação espontânea foram demonstradas para restaurar rapidamente a homeostase glutamato9,13. Portanto, não é de surpreender que a adaptação de B. subtilis a uma condição específica de crescimento durante a domesticação da bactéria seja espelhada na síntese enzimática e nas atividades enzimáticas evoluídas, que estão envolvidas no metabolismo do glutamato12. Tem sido sugerido que a falta de glutamato exógeno no meio de crescimento durante o processo de domesticação foi a força motriz para o surgimento e fixação do gene dehidroase de glutamato enigmático (GDH) gudBCR na cepa laboratorial 1682,14. Esta hipótese é apoiada pela nossa observação de que a quantidade reduzida de atividade de GDH na cepa laboratorial fornece às bactérias uma vantagem de crescimento seletiva quando o glutamato exógeno é escasso2. Além disso, o cultivo de uma cepa B. subtilis, sintetizando o GDH GudB, na ausência de glutamato exógeno resulta no acúmulo de mutantes supressores que inativaram o gene gudB 2. Obviamente, a presença de um GDH catabolicamente ativo é desvantajosa para a célula porque o glutamato produzido endógenamente que poderia ser usado para o anabbolismo é degradado para amônio e 2-oxoglutarate(Figura 1). Em contraste, quando o glutamato é fornecido pelo meio, uma cepa B. subtilis equipada com atividade GDH de alto nível tem uma vantagem de crescimento seletiva sobre uma cepa que sintetiza apenas um GDH funcional. É razoável supor que a atividade gdh de alto nível permite que as bactérias utilizem o glutamato como segunda fonte de carbono, além de outras fontes de carbono fornecidas pelo meio2 (ver Figura 1). Assim, a atividade GDH afeta fortemente o condicionamento físico das bactérias, dependendo da disponibilidade de glutamato exógeno.
Aqui apresentamos um método muito ilustrativo para monitorar e visualizar a competição intraespécie entre duas cepas de B. subtilis que diferem em um único lócus no cromossomo(Figura 2). As duas cepas foram rotuladas com os genes YFP e CFP codificando os fluoroforos YFP e CFP, e cocultivadas em diferentes condições nutricionais. Através da amostragem ao longo do tempo e emplacando diluições apropriadas em placas de ágar, os sobreviventes em cada uma das culturas poderiam ser facilmente monitorados usando um microscópio de fluorescência estéreo comum. O procedimento descrito neste artigo é fácil de realizar e adequado para visualizar a rápida expansão clonal e eliminação de mutações benéficas e prejudiciais, respectivamente, em uma população celular ao longo do tempo.

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		<tr height="26">
			<td height="26" style="height:26px;width:363px;">(NH4)2SO4&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">3746</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Agar</td>
			<td style="width:175px;">Difco, USA</td>
			<td style="width:111px;">214010</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="40">
			<td height="61" rowspan="2" style="height:61px;width:363px;">Ammonium ferric citrate (CAF)</td>
			<td rowspan="2" style="width:175px;">Sigma-Aldrich, Germany</td>
			<td rowspan="2" style="width:111px;">9714</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">CaCl2&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">5239</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Glucose</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3617</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Glycerol</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">4043</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">K2HPO4 x 3 H2O</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">6878</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">KCl</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3582</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">KH2PO4</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">3904</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">KOH</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">6751</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">MgSO4 x 7 H2O&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">P027</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">MnCl2&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">T881</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="40">
			<td height="61" rowspan="2" style="height:61px;width:363px;">MnSO4 x 4 H2O</td>
			<td rowspan="2" style="width:175px;">Merck Millipore, Germany</td>
			<td rowspan="2" style="width:111px;">102786</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">NaCl</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">9265</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Nutrient broth</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">X929</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Potassium glutamate</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3712</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Tryptone</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">8952</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Tryptophan</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3445</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Yeast extract</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">2363</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">1.5 ml Reaction tubes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">72,690,001</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">2.0 ml Reaction tubes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">72,691</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">15 ml Plastic tubes with screw cap</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">62,554,001</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Petri dishes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">82.1473</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">1.5 ml Polystyrene cuvettes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">67,742</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">15 ml Glass culture tubes&#160;</td>
			<td rowspan="2" style="width:175px;">Brand, Germany</td>
			<td rowspan="2" style="width:111px;">7790 22</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">with aluminium caps</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">100 ml Shake flasks with aluminium caps</td>
			<td style="width:175px;">Brand, Germany</td>
			<td style="width:111px;">928 24</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Sterile 10 ml glass pipettes</td>
			<td style="width:175px;">Brand, Germany</td>
			<td style="width:111px;">278 23</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Incubator (28 and 37 &#176;C)</td>
			<td style="width:175px;">New Brunswick</td>
			<td style="width:111px;">M1282-0012</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="42">
			<td height="85" rowspan="3" style="height:85px;width:363px;">Standard pipette set (2-20 &#956;l, 10-100 &#956;l, 100-1000 &#956;l)</td>
			<td rowspan="3" style="width:175px;">Eppendorf, Germany</td>
			<td style="width:111px;">4910&#160;000.034, 4910 000.042,&#160;&#160;</td>
			<td rowspan="3" style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:111px;">4910 000.042,</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:111px;">4910 000.069&#160;</td>
		</tr>
		<tr height="80">
			<td height="101" rowspan="2" style="height:101px;width:363px;">Table top centrifuge for 1.5 and 2 ml reaction tubes</td>
			<td rowspan="2" style="width:175px;">Thermo Scientific, Heraeus Fresco 21, Germany</td>
			<td rowspan="2" style="width:111px;">75002425</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="60">
			<td height="81" rowspan="2" style="height:81px;width:363px;">Table top centrifuge for 15 ml plastic tubes</td>
			<td rowspan="2" style="width:175px;">Heraeus Biofuge Primo R, Germany</td>
			<td rowspan="2" style="width:111px;">75005440</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="80">
			<td height="101" rowspan="2" style="height:101px;width:363px;">Standard spectrophotometer</td>
			<td rowspan="2" style="width:175px;">Amersham Biosciences Ultrospec 2100 pro, Germany</td>
			<td rowspan="2" style="width:111px;">80-2112-21&#160;</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Stereofluorescence microscope&#160;</td>
			<td style="width:175px;">Zeiss SteREO Lumar V12, Germany</td>
			<td style="width:111px;">495008-0009-000</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Freezer (-20 and -80 &#176;C)</td>
			<td style="width:175px;">-</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Fridge (4 &#176;C)</td>
			<td style="width:175px;">-</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Autoclave</td>
			<td style="width:175px;">Zirbus, LTA 2x3x4, Germany</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">pH meter</td>
			<td style="width:175px;">pH-meter 766, Calimatic, Knick, Germany</td>
			<td style="width:111px;">766</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="40">
			<td height="61" rowspan="2" style="height:61px;width:363px;">Vortex</td>
			<td rowspan="2" style="width:175px;">Vortex&#160; 3, IKA, Germany</td>
			<td rowspan="2" style="width:111px;">3340000</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="21">
			<td height="64" rowspan="3" style="height:64px;width:363px;">Balance</td>
			<td rowspan="3" style="width:175px;">CP2202S, Sartorius, Germany</td>
			<td style="width:111px;">replaced by</td>
			<td rowspan="3" style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:111px;">CPA2202S</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:111px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Black pen (permanent marker)</td>
			<td style="width:175px;">Staedler, Germany</td>
			<td style="width:111px;">317-9</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Powerpoint program</td>
			<td style="width:175px;">Microsoft, USA</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Office Excel program</td>
			<td style="width:175px;">Microsoft, USA</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">Program for data processing</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Adobe Photoshop CS5</td>
			<td style="width:175px;">Adobe, USA</td>
			<td style="width:111px;">replaced by CS6, download</td>
			<td style="width:167px;">Computer program for image processing</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Computer</td>
			<td style="width:175px;">PC or Mac</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">ZEN pro 2011 software for the stereofluorescence microscope</td>
			<td style="width:175px;">Zeiss, Germany</td>
			<td style="width:111px;">410135 1002 110</td>
			<td style="width:167px;">AxioCam MRc Rev. Obtained through Zeiss</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:815px;">Specific solution recipes</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">SP medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">8 g Nutrient broth</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">0.25 mg MgSO4 x 7 H2O</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">1 g KCl</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">if required, add 15 g agar for solid SP medium</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">1 ml CaCl2 (0.5 M), sterilized by filtration</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">1 ml MnCl2 (10 mM) sterilized by filtration</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:815px;">2 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">LB medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 g Tryptone</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">5 g Yeast extract</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 g NaCl</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">if required, add 15 g agar for solid LB medium</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">C-Glc minimal medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">200 ml 5 x C salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml L-Tryptophan (5 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml III&#8217; salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">25 ml Glucose (20%), autoclaved for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">CE-Glc minimal medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">200 ml 5 x C salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml L-Tryptophan (5 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml III&#8217; salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">20 ml Glutamate (40%)</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">25 ml Glucose (20%), autoclaved for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">5 x C salts&#160;</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">20 g KH2PO4</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">80 g K2HPO4 x 3 H2O</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">16.5 g (NH4)2SO4</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">III&#8217; salts</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">0.232 g MnSO4 x 4 H2O</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">12.3 g MgSO4 x 7 H2O</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">40% Glutamate solution</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">200 g L-Glutamic acid</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">adjust the pH to 7.0 by adding approximately 80 g KOH</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 0.5 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">0.9% Saline (NaCl) solution</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">50% Glycerol solution</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">295 ml Glycerol (87%)</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 0.5 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:815px;">Bacteria (All strains are based on the Bacillus subtilis strain 168)</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP40 (rocG+ gudBCR amyE::PgudB-yfp)&#160;</td>
			<td rowspan="4" style="width:167px;">Laboratory strain collection</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP41 (rocG+ gudBCR amyE::PgudB-cfp)&#160;</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP52 (rocG+ gudB+ amyE::PgudB-cfp)</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP156 (rocG+ gudB+ amyE::PgudB-yfp)</td>
		</tr>
	</tbody>
</table>

monitoring intraspecies competition in a bacterial cell population by cocultivation of fluorescently labelled strains

Muitos microrganismos, como bactérias, proliferam extremamente rápido e as populações podem atingir altas densidades celulares. Pequenas frações de células em uma população sempre têm mutações acumuladas que são prejudiciais ou benéficas para a célula. Se o efeito fitness de uma mutação fornece a subpopulação com uma forte vantagem de crescimento seletivo, os indivíduos desta subpopulação podem rapidamente superar e até eliminar completamente seus companheiros imediatos. Assim, pequenas alterações genéticas e acúmulo de células que adquiriram mutações benéficas podem levar a uma mudança completa do genótipo de uma população celular. Aqui apresentamos um procedimento para monitorar a rápida expansão clonal e a eliminação de mutações benéficas e prejudiciais, respectivamente, em uma população celular bacteriana ao longo do tempo por cocultivação de indivíduos fluorescentes rotulados da bactéria modelo Gram-positivo Bacillus subtilis. O método é fácil de executar e muito ilustrativo para exibir competição intraespécie entre os indivíduos em uma população celular bacteriana.

O método aqui descrito foi aplicado com sucesso para visualizar a competição de intraespécies em uma população celular constituída por cepas de B. subtilis que foram rotuladas com os genes CFP e YFP codificando os fluoroforos CFP e YFP, respectivamente. Como mostrado na Figura 3,o método pode ser usado para visualizar a competição de intraespécies de forma muito ilustrativa. Ao detectar as amostras em pequenas áreas, a composição clonal da população celular torn...

Watch this Scientific Journal Video about Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains at JoVE.com

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

As bactérias podem acumular mutações prejudiciais ou benéficas durante a vida. Em uma população de células indivíduos que acumularam mutações benéficas podem rapidamente superar seus companheiros. Aqui apresentamos um procedimento simples para visualizar a competição de intraespécies em uma população de células bacterianas ao longo do tempo usando indivíduos rotulados fluorescentes.

Monitorando a competição de subespécies em uma população de células bacterianas por cocultivação de cepas fluorescentes rotuladas

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a ...

monitoring-intraspecies-competition-bacterial-cell-population

Research

JoVE Journal

Biology

8.4K visualizações.  Georg-August University. As bactérias podem acumular mutações prejudiciais ou benéficas durante a vida. Em uma população de células indivíduos que acumularam mutações benéficas podem rapidamente superar seus companheiros. Aqui apresentamos um procedimento simples para visualizar a competição de intraespécies em uma população de células bacterianas ao longo do tempo usando indivíduos rotulados fluorescentes.

Vídeo: Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Title Cell Encapsulation by Droplets

Encapsulation título celular por Droplets

Biologia

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivação de células estaminais embrionárias humanas por Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Mudanças Dinâmicas de monitoramento dos níveis de cálcio mitocondrial durante a apoptose através de um sensor de cálcio geneticamente codificado

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Ativação da apoptose por microinjeção citoplasmática de citocromo C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

Atividades de monitoramento quinases e fosfatases através do ciclo celular por raciométrica FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking1,2 but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases3.
Yet their existence is still a matter of controversy4,5. Indeed, lipid raft size has been estimated to be around 20 nm6, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized7,8. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells.
Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts9,10. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models11.
FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently12. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change12.

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS

A technique to probe the lipid raft partitioning of fluorescent proteins at the plasma membrane of living cells is described. It takes advantage of the disparity in diffusion times of proteins located inside or outside of lipid rafts. Acquisition can be performed dynamically in control conditions or after drug addition.

Determinação da Raft Lipid Particionamento de Sondas marcadas com fluorescência em células vivas por Espectroscopia de Correlação de Fluorescência (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A longo prazo de Teste de toxicidade letal com o crustáceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Profiling Individual células estaminais embrionárias humanas por RT-PCR quantitativo

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal Análise de glicocorticóides: Non-invasive Monitoring Adrenal em eqüídeos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and product development. Stress-strain relationships are typically obtained from mechanical testing data to enable comparative assessment among samples and in some cases identification of constitutive mechanical properties. However, errors may be introduced through the use of average strain measures, as significant heterogeneity in the strain field may result from geometrical non-uniformity of the sample and stress concentrations induced by mounting/gripping of soft tissues within the test system. When strain field heterogeneity is significant, accurate assessment of the sample mechanical response requires measurement of local strains. This study demonstrates a novel biomechanical testing protocol for calculating local surface strains using a mechanical testing device coupled with a high resolution camera and a digital image correlation technique. A series of sample surface images are acquired and then analyzed to quantify the local surface strain of a vascular tissue specimen subjected to ramped uniaxial loading. This approach can improve accuracy in experimental vascular biomechanics and has potential for broader use among other native soft tissues, engineered soft tissues, and soft hydrogel/polymeric materials. In the video, we demonstrate how to set up the system components and perform a complete experiment on native vascular tissue.

We describe the use of digital image correlation to characterize the local surface strain field on vascular tissue samples subjected to uniaxial tensile testing. These measurements facilitate precise quantification of the sample mechanical response and the generation of constitutive stress-strain relations.

Usando Correlação Digital de Imagem para caracterizar as cepas de locais vasculares amostras de tecido

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and ...