Este estudo foi financiado pelo National Heart, Lung, and Blood Institute Grant HL- 103542, e Distinguished Trailblazer Award O BCH da Fundação Anesthesia Research para CAP.

Preparação <ol><li> Prepare 1 M K-HEPES da Solução (ajustar o pH para 7,2 com KOH). </li><li> Prepare 0,5 M K-EGTA da Solução (ajustar o pH para 8,0 com KOH). </li><li> Prepare 1 M KH 2 PO 4 da solução. </li><li> Prepare 1 M MgCl2 da solução. </li><li> Preparar tampão de homogeneização (pH 7,2) 300 mM de sacarose, 10 mM de K-HEPES, e 1 mM de K-EGTA. Armazenar a 4 ° C. </li><li> Preparar tampão de respiração de 250 mM de sacarose, 2 mM de KH 2 PO 4, 10 mM de MgCl2, 20 mM de K-HEPES (pH 7,2) e 0,5 mM de K-EGTA (pH 8,0). Armazenar a 4 ° C. </li><li> Prepare PBS 10x da solução por dissolução de 80 g de NaCl, 2 g de KCl, 14,4 g de Na 2 HPO 4, e 2,4 g de KH 2 PO 4 em 1 L duplo H2O destilada (pH 7,4). </li><li> Prepare 1x PBS por pipetagem de 100 ml de 10x PBS em 1 L duplo H2O destilada </li><li> Prepare a Subtilisina A Stock por pesagem de 4 mg de Subtilisina A empara um tubo de microcentrífuga de 1,5 ml. Armazenar a -20 ° C até o uso. </li><li> Preparar por pesagem da BSA a 20 mg de BSA para um tubo de microcentrífuga de 1,5 ml. Armazenar a -20 ° C até o uso. </li></ol> Isolamento mitocondrial (Figura 1) <ol><li> Imediatamente antes do isolamento, dissolver Subtilisina A em 1 ml de tampão de homogeneização. </li><li> Imediatamente antes do isolamento, dissolver de BSA em 1 ml de tampão de homogeneização. </li><li> Recolher duas amostras de tecido fresco utilizando um perfurador 6 milímetros amostra de biópsia e armazenar em 1x PBS num tubo de centrífuga cónico de 50 ml em gelo. </li><li> Transferir os dois perfuradores 6 mm de tecido para um tubo de dissociação C contendo 5 ml de tampão de homogeneização arrefecida com gelo. </li><li> Homogeneizar o tecido, ajustando o tubo de dissociação C no Dissociator tecido e selecione a pré-definido ciclo isolamento mitocondrial (60 seg homogeneização). </li><li> Retire o tubo de dissociação C para um balde de gelo. </li><li> Adicionar 250 mL de uma solução de estoque Subtilisin aohomogeneizado, misturar por inversão e incubar o homogeneizado em gelo durante 10 min. </li><li> Colocar um filtro de malha de 40 um para um tubo de centrífuga cónico de 50 ml em gelo, pré-molhada com o filtro de tampão de homogeneização e filtrar o homogenato para o tubo de centrífuga cónico de 50 ml em gelo. </li><li> Adicionar 250 ul de BSA preparada de fresco da Solução ao filtrado e misturar por inversão. Nota: Ignore essa etapa se a determinação da proteína mitocondrial é necessária. </li><li> Colocar um filtro de 40 um para um tubo de centrífuga cónico de 50 ml em gelo, pré-molhada com o filtro de tampão de homogeneização e filtrar o homogenato para o tubo de centrífuga cónico de 50 ml em gelo. </li><li> Colocar um filtro de 10 um para um tubo de centrífuga cónico de 50 ml em gelo, e pré-molhada com o filtro de tampão de homogeneização e filtrar o homogenato para o tubo de centrífuga cónico de 50 ml em gelo. </li><li> Transferir o filtrado para dois pré-refrigerada tubos de 1,5 ml de microcentrífuga e centrifugue a 9000 xg durante 10 min a4 ° C. </li><li> Remover o sobrenadante e re-suspender e combinar pelotas em 1 ml de gelo frio Respiração Buffer. </li></ol> ATP Assay Nota: Para determinar a actividade metabólica da mitocôndria isolada de um ensaio de luminescência de ATP pode ser realizada utilizando um kit de ensaio de ATP. O protocolo, e os reagentes foram os padrões fornecidos no kit de ensaio. Um resumo do processo é descrito abaixo. <ol><li> Equilibrar reagentes para RT. </li><li> Preparar 10 mM de ATP da solução através da dissolução de ATP sedimento liofilizado em 1170 mL de água duplamente destilada. Guarde ATP padrão da solução e amostras mitocondriais preparadas no gelo. </li><li> Adicionar 5 ml de solução de tampão de substrato para um frasco de solução de substrato liofilizado. Misture delicadamente e coloque no escuro. </li><li> Adicionar 100 ul de tampão de respiração de todos os poços de uma preta, de fundo opaco, placa de 96 poços. </li><li> Adicionar 10 ul de mitocôndrias das amostras preparadas a cada poço ofa preta, de fundo opaco, placa de 96 poços. Nota: As amostras são colocadas em placas em triplicado. Inclua uma linha para os padrões e três poços para o controle negativo (Respiração Buffer). </li><li> Adicionar 50 ul de solução de lise de células de mamíferos a todos os poços, incluindo padrões e controlos. </li><li> Incubar a placa a 37 ° C por 5 min em um agitador orbital a 125 rpm. </li><li> Durante a incubação preparar padrões de ATP em concentrações de 0,1 mM, 0,05 mM, 0,01 mM, 0,005 mM, 0,001 mM e 0,0001 mM de ATP a partir de ATP 10 mM da solução. Padrão das lojas no gelo. </li><li> Após a incubação, adicionar 10 ul de ATP padrão aos poços correspondentes como indicado na placa de mapa. Nota: Os padrões são realizadas em duplicata. </li><li> Adicionar 50 ul da solução de substrato a cada poço reconstituído. </li><li> Incubar a placa a 37 ° C num agitador orbital durante 5 minutos a 125 rpm. </li><li> Abra o software Gen5 1.11 em um computador ligado com o espectrofotômetro. </li><li> Em &quot;Criar um novo item &quot;, clique em&quot; Experiment &quot;. </li><li> Clique em &quot;protocolo padrão&quot;. Clique em &quot;Ok&quot;. </li><li> Na coluna da esquerda, selecione &quot;Protocolo&quot;, depois em &quot;Procedimento&quot;. </li><li> Selecione &quot;Delay&quot;. Defina a 0:10:00. Clique em &quot;Ok&quot;. </li><li> Selecione &quot;Ler&quot;. Selecione &quot;luminescência&quot; do menu suspenso. Ajuste outras definições para o seguinte: Leia tipo Endpoint, Tempo de Integração 0: 01.00 MM: ss.ss, Filter Sets 1, Emissão Hole, Ótica posição, Sensibilidade 100, Top Probe Desvio vertical 1,00 milímetros. </li><li> Quando a placa está pronta para ser analisada, clique no ícone &quot;Leia Plate&quot; na linha superior. Clique em &quot;Read&quot;. Quando o espectrofotômetro abrir, colocar a placa na bandeja com poço A1 no canto superior esquerdo. Uma caixa com a temperatura vai abrir. Clique em &quot;Read&quot;. Nota: Os valores mais elevados correlacionam-se com os níveis de ATP aumento e maior atividade metabólica. </li></ol>

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Todos os animais foram tratados de acordo com as diretrizes institucionais vigentes. Não temos interesses financeiros competitivos para divulgar.

Para isolar com sucesso mitocôndrias que utilizam este protocolo é fundamental para manter todas as soluções e amostras de tecido em gelo para preservar a viabilidade mitocondrial. Mesmo quando mantidos em gelo, as mitocôndrias isoladas irá exibir uma diminuição na actividade funcional ao longo do tempo 19. Recomendamos que todas as soluções e adições ser pré-preparado. Nós pré-pesar e armazenar Subtilisina A em alíquotas de 4 mg em 1,5 ml de tubos de microcentrífuga e armazená-las a -20 ° ...

A correction was made to: <a href="http://www.jove.com/video/51682">Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration.</a> An optional step was added to step 7 of the &#39;Mitochondrial Isolation&#39; section of the protocol. The step is:
1. Optional in the case that the tissue is fibrous: Centrifuge the solution at 750 x g for 4 min.

A correction was made to: Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration.

Erratum: Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

As mitocôndrias existem em todas as células do corpo, excepto as células vermelhas do sangue e são envolvidos num grande número de importantes processos celulares e metabólicas 1-4. Devido a estas várias funções, dano mitocondrial pode ter efeitos prejudiciais 3. A fim de investigar a função mitocondrial e disfunção de vários métodos de isolamento mitocondriais foram descritos. Os primeiros relatos publicados de data isolamento mitocondrial à década de 1940 5-8. A primeira tentativa documentada demonstrado isolamento mitocondrial por trituração do tecido do fígado, num almofariz, seguido por centrifugação de uma solução de sal de 5,8 a baixa velocidade. Mais tarde, outros grupos expandiu o procedimento original e demonstrou fraccionamento de tecido com base na centrifugação diferencial 6-8. Estes métodos iniciais formaram a base das técnicas atuais que muitas vezes incorporam homogeneização e / ou centrifugação diferencial 9-15. O número de homogenizatiem passos de centrifugação e varia entre protocolos. Estes passos repetitivos aumentar o tempo para o isolamento mitocondrial e, finalmente, reduzir a viabilidade. Além disso, a homogeneização manual pode causar danos e inconsistentes resultados mitocondriais se não for devidamente controlado 10,16. Recentemente, foi utilizado homogeneização e centrifugação diferencial para isolar mitocôndrias para transplante no tecido do miocárdio 17,18. Este procedimento de isolamento demorado necessário cerca de 90 min e a aplicabilidade clínica deste método era, portanto, limitado. Para possibilitar a utilização terapêutica aguda em tratamentos clínicos e cirúrgicos, desenvolvemos um procedimento de isolamento mitocondrial rápido que pode ser realizada em menos de 30 min. Os principais benefícios deste protocolo são de que a dissociação do tecido padronizado permite uniforme e consistente homogeneização do tecido que não é facilmente alcançada com homogeneização manual. Em additião, o uso de filtração diferencial em lugar de centrifugação diferencial elimina demorado e passos de centrifugação repetitivas que permitam o isolamento de mitocôndrias mais rápida competentes altamente purificadas, viáveis ​​e respiratória. A capacidade de isolar mitocôndrias competentes viáveis ​​e de respiração, em menos de 30 min permite a aplicabilidade clínica. Este protocolo de isolamento tem potencial para uso em cirurgia de revascularização do miocárdio (CRM) e outros procedimentos terapêuticos.

<table border="0" cellpadding="0" cellspacing="0" width="933">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:377px;">Sucrose</td>
			<td style="width:181px;">Sigma Aldrich</td>
			<td style="width:147px;">84100</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Substilsin A</td>
			<td>Sigma Aldrich</td>
			<td>P5380</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BSA</td>
			<td>Sigma Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KH2PO4</td>
			<td>Sigma Aldrich</td>
			<td>P5379</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MgCl2</td>
			<td>Sigma Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S6191</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>Fisher Scientific</td>
			<td>P2173</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>S374</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:377px;">ATPlite Luminescence Assay System, 
			1000 Assay Kit</td>
			<td>Perkin Elmer</td>
			<td>6016941</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 mL Conical Tubes</td>
			<td>BD</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40 &#956;m Nylon Filters</td>
			<td>BD&#160;</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GentleMACS C tube</td>
			<td>Miltenyl Biotech</td>
			<td>120-005-331</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 mL Eppendorf tube</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6 mm biopsy punch</td>
			<td>Miltex</td>
			<td>33-36</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 &#956;m Pluristrainer</td>
			<td>Pluriselect</td>
			<td>43-500-10-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorf Centrifuge 5415C</td>
			<td>Marshall Scientific</td>
			<td>EP-5415C&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GentleMACS Dissociator&#160;</td>
			<td>Miltenyl Biotech</td>
			<td>130-093-235</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-well plates, tissue culture treated</td>
			<td>VWR</td>
			<td>82050-732</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rotomax 120 orbital shaker</td>
			<td>Heidolph</td>
			<td>544-41200-00</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Synergy H4 Hybrid Multi-Mode 
			&#160;Microplate Reader</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Multisizer 4 Coulter Counter</td>
			<td>Beckman Coulter</td>
			<td>A63076</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oxytherm System</td>
			<td>Hansatech Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hemacytometer</td>
			<td>Fisher Scientific</td>
			<td>267110</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid isolation and purification of mitochondria for transplantation by tissue dissociation and differential filtration

Métodos de isolamento mitocondriais descritas anteriormente, utilizando centrifugação diferencial e / ou centrifugação em gradiente de Ficoll exigir de 60 a 100 min para completar. Descreve-se um método para o isolamento rápido de mitocôndrias a partir de biópsias de mamífero, utilizando um tecido comercial Dissociator filtração e diferencial. Neste protocolo, homogeneização manual é substituído por ciclo de homogeneização padronizada do Dissociator tecido. Isto permite uma uniforme e consistente homogeneização de tecido que não é facilmente conseguido com a homogeneização manual. Após a dissociação de tecidos, o produto homogeneizado é filtrado através de filtros de nylon de malha, o que elimina os passos de centrifugação repetitivas. Como resultado, o isolamento mitocondrial pode ser realizada em menos de 30 min. Este protocolo de isolamento rende aproximadamente 2 x 10 10 mitocôndrias competentes viáveis ​​e respiratória de 0,18 ± 0,04 g (peso húmido) de uma amostra de tecido.

A figura delinear os passos processuais no isolamento de mitocôndrias utilizando dissociação do tecido e filtração diferencial está representada na Figura 1. Tempo processual total é inferior a 30 min. As amostras de tecido foram obtidas utilizando um punch de 6 mm. O peso do tecido era de 0,18 ± 0,04 g (peso molhado). O número de mitocôndrias isoladas, como determinado por contagem de tamanho de partícula foi de 2,4 x 10 10 ± 0,1 x 10 10 mi...

Watch this Scientific Journal Video about Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration at JoVE.com

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Um método para o isolamento rápido de mitocôndrias a partir de biópsias de tecidos de mamíferos é descrito. Preparações de fígado de rato ou de músculo esquelético foram homogeneizados com um Dissociator tecido comercial e mitocôndrias foram isoladas por meio de filtração através de filtros de diferencial de malha de nylon. Tempo de isolamento mitocondrial é &lt;30 min, em comparação com 60 - 100 min utilizando métodos alternativos.

Rápido isolamento e purificação de mitocôndrias para o transplante pela dissociação do tecido e Filtração Diferencial

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

rapid-isolation-purification-mitochondria-for-transplantation-tissue

Research

JoVE Journal

Biology

37.7K visualizações.  Beth Israel Deaconess Medical Center and Harvard Medical School.  Um método para o isolamento rápido de mitocôndrias a partir de biópsias de tecidos de mamíferos é descrito. Preparações de fígado de rato ou de músculo esquelético foram homogeneizados com um Dissociator tecido comercial e mitocôndrias foram isoladas por meio de filtração através de filtros de diferencial de malha de nylon. Tempo de isolamento mitocondrial é 

Vídeo: Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

Isolamento e Transplante de Células-Tronco Hematopoéticas (HSCs)

Biologia

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Coloração de proteínas em géis com Coomassie G-250 sem ácido acético e Solventes Orgânicos

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

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 ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Isolamento e purificação de Drosophila Neurônios periféricos por classificação Bead Magnética

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Isolamento de cromatina por purificação de RNA (CHIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

Imobilização planária, irradiação parcial e Transplante de Tecidos

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Isolamento, cultura e transplante de células musculares satélite

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Isolamento e Análise Funcional de mitocôndrias de células cultivadas e mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

Isolamento de filtração de Ácidos Nucleicos: Um Método de extracção de DNA simples e rápido

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.