Este trabalho foi financiado por concessões do CIHR e NSERC do Canadá. VIT é um CIHR New Investigator e Concordia University, em Cátedra de Investigação Genómica, Biologia Celular e Envelhecimento.

Materiais e métodos Cepas de leveduras e condições de crescimento A cepa do tipo selvagem BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) foi cultivado em meio YEPD rica (1% extrato de levedura, peptona 2%, 2% de glicose). Células foram cultivadas a 30 ° C com rotação agitação a 200 rpm em Erlenmeyers em um &quot;volume frasco de volume / médio&quot; proporção de 5:1. Isolamento da fração mitocondrial crude <ol><li> Crescer 1 L da cultura BY4742 tipo selvagem tensão por 48 h. </li><li> Despeje cultura em 500 ml tubos de centrífuga Nalgene. Equilibrar a carga e células de colheita por centrifugação por 5 min a 3000 xg à temperatura ambiente. </li><li> Decantar o sobrenadante e ressuspender as células peletizadas em 250 ml de dH2O. Pellet células por centrifugação por 5 min a 3000 xg à temperatura ambiente. </li><li> Decantar o sobrenadante e ressuspender as células peletizadas em 250 ml de dH2O. Pellet células por centrifugação por 5 min a 3000 xg à temperatura ambiente. </li><li> Determinar o peso úmido de pellet celular. </li><li> Ressuspender as células peletizada na TDT buffer [2 ml de células (peso úmido) buffer / g]. </li><li> Transferir a suspensão de célula para 50 ml, tubos de plástico Falcon. </li><li> Girar os tubos a 70 rpm em um agitador por 20 min a 30 ° C. </li><li> Células colheita por centrifugação por 5 min a 3000 xg à temperatura ambiente. </li><li> Ressuspender as células em tampão Zymolyase peletizadas sem Zymolyase [7 ml de células (peso úmido) buffer / g]. </li><li> Pellet células por centrifugação por 5 min a 3000 xg à temperatura ambiente. </li><li> Ressuspender as células em tampão Zymolyase peletizadas sem Zymolyase [7 ml de células (peso úmido) buffer / g]. </li><li> Transferência de suspensão de células para um balão de vidro. </li><li> Adicionar o pó de Zymolyase-100T [1 mg de células (peso úmido) Zymolyase-100T / g] para a suspensão de células. </li><li> Gire o frasco com suspensão de células em 70 rpm em um agitador por 30 min a 30 ° C. </li><li> Esferoplastos transferência formado devido à digestão da parede celular com Zymolyase-100T de 50 ml, tubos de centrífuga de plástico. </li><li> Pellet esferoplastos por centrifugação por 8 min a 2200 xg, a 4 ° C. </li></ol> ** Todas as etapas subseqüentes devem ser realizados a 4 ° C ou em gelo. Suspensões de esferoplastos devem ser manuseados com cuidado usando pipetas com pontas cortadas para evitar a quebra organelas .** <ol start="18"><li> Ressuspender esferoplastos peletizada na gelada tampão de homogeneização [6,5 ml de células (peso úmido) buffer / g]. </li><li> Pellet esferoplastos por centrifugação por 8 min a 2200 xg, a 4 ° C. </li><li> Ressuspender esferoplastos peletizada na gelada tampão de homogeneização [6,5 ml de células (peso úmido) buffer / g]. </li><li> Transferência de células para um pré-refrigeradas em gelo copo homogeneizador. </li><li> Usando um pilão apertado, homogeneizar as células, fazendo 15 batidas do pilão. </li><li> Adicionar um volume de gelada tampão de homogeneização. </li><li> Transferência esferoplastos homogeneizada a 50 ml tubos de centrífuga de plástico. </li><li> Pellet as células intactas, núcleos e partículas grandes por centrifugação por 5 min a 1500 xg, a 4 ° C. </li><li> Centrifugar o sobrenadante resultante para 5 min a 3000 xg, a 4 ° C. </li><li> Centrifugar o sobrenadante por 15 min a 12000 xg, a 4 ° C. </li><li> Decantar o sobrenadante e ressuspender em pellet gelada tampão de homogeneização [6,5 ml de células (peso úmido) buffer / g]. </li><li> Centrifugar por 5 min a 3000 xg, a 4 ° C. </li><li> Centrifugar o sobrenadante por 15 min a 12000 xg, a 4 ° C. </li><li> Decantar o sobrenadante e ressuspender pellet em 3 ml de gelado de buffer SEM. </li></ol> ** Embora esta suspensão é enriquecido na mitocôndria, que também contém outras organelas como o retículo endoplasmático (microssomos), Golgi e vacúolos. Para obter a mitocôndria pura, esta fração de petróleo mitocondrial pode ser submetido a fracionamento ainda, conforme descrito abaixo .** Purificação de mitocôndrias desprovida de contaminação por outras organelas <ol><li> Ml lugar de 1,5 gelada 60% (w / v) de sacarose na EM tampão em um tubo de centrífuga Beckman Ultra-Clear. </li><li> Sobreposição de 60% (w / v) de sacarose, com 4 ml de 32%, 1,5 ml de 23%, 1,5 ml de sacarose 15% (todos tampão wt / v em EM). </li><li> Coloque 3 ml da fração mitocondrial em bruto SEM tampão em cima de 15% (w / v) de sacarose. </li><li> Centrífuga em uma Ti SW41 rotor Beckman balançando-balde por 1 hora a 134.000 xg (33.000 rpm) a 4 ° C. </li><li> A mitocôndria forma intacta uma faixa marrom na interface de sacarose 60% / 32%. </li><li> Remova cuidadosamente sacarose até chegar a banda mitocondrial. </li><li> Retire a banda mitocondrial utilizando uma pipeta com uma ponta de corte e colocá-lo em um tubo de centrífuga Beckman para um rotor-5 MLS. </li><li> Encha o tubo com tampão de SEM. </li><li> Pellet as mitocôndrias pura por centrifugação em um rotor MLS-5 para 30 min a 10.000 xg (31.000 rpm) durante 30 minutos a 4 ° C. </li><li> Decantar o sobrenadante usandouma pipeta. </li><li> Use as mitocôndrias pura para a análise de funções mitocondriais, proteoma mitocondrial e lipidome e DNA mitocondrial. </li></ol> Reagentes <ol><li> TDT buffer [100 mM Tris/H2SO4 (pH 9,4), 10 mM ditiotreitol] (para 15 ml) <ul style="list-style-type:none;"><li> 1,5 ml de um tampão Tris/H2SO4 M (pH 9,4) </li><li> 150 mL de 1 M DTT </li><li> Volume para 15 ml </li></ul></li><li> Zymolyase tampão [20 mM fosfato de potássio (pH 7,4), 1,2 M sorbitol] (para 100 ml) <ul style="list-style-type:none;"><li> 2 ml de um tampão fosfato de potássio M (pH 7,4) </li><li> 60 ml de 2 M sorbitol </li><li> Volume de 100 ml </li></ul></li><li> Tampão de homogeneização [10 mM Tris / HCl (pH 7,4), 0,6 M sorbitol, 1 mM EDTA, 0,2% (w / v) BSA] (para 250 ml] <ul style="list-style-type:none;"><li> 2,5 ml de 1 M Tris / HCl (pH 7,4) </li><li> 75 ml de 2 M sorbitol </li><li> 500 mL de 500 mM EDTA </li><li> 0,5 g de BSA </li><li> Volume para 250 ml </li></ul></li><li> SEM buffer [10 mM MOPS / KOH (pH 7,2), 250 mM de sacarose, 1 mM EDTA] (para 250 ml) <ul style="list-style-type:none;"><li> 2,5 ml de 1 MOPS M / buffer KOH (pH 7,2) </li><li> 31,25 ml de 2 M de sacarose </li><li> 500 mL de 500 mM EDTA </li><li> Volume para 250 ml </li></ul></li><li> EM buffer [10 mM MOPS / KOH (pH 7,2), 1 mM EDTA] (para 250 ml) <ul style="list-style-type:none;"><li> 2,5 ml de 1 MOPS M / buffer KOH (pH 7,2) </li><li> 500 mL de 500 mM EDTA </li><li> Volume para 250 ml </li></ul></li></ol>

<ol>
	<li>Voet, D., Voet, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry.">Biochemistry.</a> , (2004).</li><li>Suen, D. -. F., Norris, K. L., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dynamics+and+apoptosis.">Mitochondrial dynamics and apoptosis.</a> Genes Dev. 22, 1577-1590 (2008).</li><li>Cheng, W. C., Berman, S. B., Ivanovska, I., Jonas, E. A., Lee, S. J., Chen, Y., Kaczmarek, L. K., Pineda, F. &. a. m. p. ;. a. m. p., Hardwick, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+factors+with+dual+roles+in+death+and+survival.">Mitochondrial factors with dual roles in death and survival.</a> Oncogene. 25, 4697-4705 (2006).</li><li>Detmer, S. A., Chan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+and+dysfunctions+of+mitochondrial+dynamics.">Functions and dysfunctions of mitochondrial dynamics.</a> Nat. Rev. Mol. Cell Biol. 8, 870-879 (2007).</li><li>McBride, H. M., Neuspiel, M., Wasiak, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+more+than+just+a+powerhouse.">Mitochondria: more than just a powerhouse.</a> Curr. Biol. 16, R551-R560 (2006).</li><li>Balaban, R. S., Nemoto, S., Finkel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria,+oxidants,+and+aging.">Mitochondria, oxidants, and aging.</a> Cell. 120, 483-495 (2005).</li></ol>

The authors have nothing to disclose.

Este método permite o isolamento de alto rendimento de mitocôndrias puro de células de levedura. Mitocôndrias purificadas por esse método são essencialmente livres de contaminação por outras organelas e manter sua integridade estrutural e funcional após a sua purificação. O método descrito rendimentos mitocôndrias, que são adequados para células livres reconstituição de processos essenciais mitocondrial. Estas mitocôndrias altamente puro pode também ser usado para a análise da estrutura mitocondrial ...

purification of mitochondria from yeast cells

As mitocôndrias são o principal local de produção de ATP durante o metabolismo aeróbico em eucariotas não fotossintéticos células<sup&gt; 1</sup&gt;. Essas organelas complexo também desempenham um papel essencial na morte celular por apoptose<sup&gt; 2</sup&gt; Sobrevivência da célula,<sup&gt; 3</sup&gt; Desenvolvimento, mamíferos<sup&gt; 4</sup&gt; Desenvolvimento, e função neuronal<sup&gt; 4</sup&gt; Sinalização, intracelular<sup&gt; 5</sup&gt;, Longevidade e regulação<sup&gt; 6</sup&gt;. Nosso entendimento desses processos biológicos complexos controlados pela mitocôndria depende métodos robustos para avaliar sua morfologia, sua proteína e composição lipídica, a integridade de seu DNA, e suas numerosas funções vitais. A levedura de brotamento<em&gt; Saccharomyces cerevisiae</em&gt;, Uma geneticamente e bioquimicamente eukaryote manipuláveis ​​unicelulares com genoma anotado e bem definidos, proteoma, é um modelo valioso para estudar os mecanismos moleculares e celulares subjacentes funções biológicas essenciais da mitocôndria. Para estes tipos de estudos, é crucial ter mitocôndrias de alta pureza. Aqui nós apresentamos uma descrição detalhada de um método rápido e eficaz para a purificação de levedura mitocôndrias. Este método permite o isolamento de mitocôndrias altamente puros que são essencialmente livres de contaminação por outras organelas e manter sua integridade estrutural e funcional após a sua purificação. Mitocôndrias purificadas por esse método são adequados para células livres reconstituição de processos essenciais mitocondrial e pode ser usado para a análise da estrutura mitocondrial e funções, proteoma mitocondrial e lipidome e DNA mitocondrial.

Watch this Scientific Journal Video about Purification of Mitochondria from Yeast Cells at JoVE.com

Purification of Mitochondria from Yeast Cells

Nós descrevemos um método rápido e eficaz para a purificação de mitocôndrias da levedura<em&gt; Saccharomyces cerevisiae</em&gt;. Este método permite o isolamento de alto rendimento de mitocôndrias puro que são essencialmente livres de contaminação por outras organelas e manter sua integridade estrutural e funcional após a sua purificação.

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 ...

purification-of-mitochondria-from-yeast-cells

Research

JoVE Journal

Biology

25.0K visualizações.  Concordia University. Nós descrevemos um método rápido e eficaz para a purificação de mitocôndrias da levedura Saccharomyces cerevisiae. Este método permite o isolamento de alto rendimento de mitocôndrias puro que são essencialmente livres de contaminação por outras organelas e manter sua integridade estrutural e funcional após a sua purificação.

Vídeo: Purification of Mitochondria from Yeast Cells

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolamento de células T CD4 + a partir dos nós linfáticos rato Usando Miltenyi Purificação MACS

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

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 Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Purificação de Progenitores do músculo esquelético

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Purificação cromatográfica de Ribossomos Yeast Highly Active

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Imagem confocal de Solteiro mitocondriais superóxido Pisca em coração intacto ou<em&gt; In Vivo</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes&#8217; association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions&#160;in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

Isolamento de mRNAs Associated com mitocôndrias de levedura para estudar mecanismos de regionalização de Tradução

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

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

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

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 ...

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 ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

A purificação de todo o genoma de DNA circular Extracromossômica de células eucarióticas

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found responsible for very diverse diseases, ranging from neurological pathologies to cancer. Interestingly, mitochondria have recently been shown to display the capacity to be transferred between cell types, notably from human mesenchymal stem cells (MSC) to cancer cells in coculture conditions, with metabolic and functional consequences for the mitochondria recipient cells, further enhancing the current interest for the biological properties of these organelles.
Evaluating the effects of the transferred MSC mitochondria in the target cells is of primary importance to understand the biological outcome of such cell-cell interactions. The MitoCeption protocol described here allows the transfer of the mitochondria isolated beforehand from the donor cells to the target cells, using MSC mitochondria and glioblastoma stem cells (GSC) as a model system. This protocol has previously been used to transfer mitochondria, isolated from MSCs, to adherent MDA-MB-231 cancer cells. This mitochondria transfer protocol is adapted here for GSCs that present the specific particularity of growing as neurospheres in vitro. The transfer of the isolated mitochondria can be followed by fluorescence-activated cell sorting (FACS) and confocal imaging using mitochondria vital dyes. The use of mitochondria donor and target cells with distinct haplotypes (SNPs) also allows detection of the transferred mitochondria based on the concentration of their circular mitochondrial DNA (mtDNA) in the target cells. Once the protocol has been validated with these criteria, the cells harboring the transferred mitochondria can be further analyzed to determine the effects of the exogenous mitochondria on biological properties such as cell metabolism, plasticity, proliferation and response to therapy.

Here, a protocol (MitoCeption) is presented to transfer mitochondria, isolated from human mesenchymal stem cells (MSC), to glioblastoma stem cells (GSC), with the goal of studying their biological effects on GSC metabolism and functions. A similar protocol can be adapted to transfer mitochondria between other cell types.

Purificação de mitocôndrias de células de levedura

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