Este trabajo fue apoyado por becas de los CIHR y NSERC de Canadá. VIT es un investigador de Nueva CIHR y Concordia Cátedra de Investigación de la Universidad de genómica, biología celular y el envejecimiento.

Materiales y métodos Cepas de levadura y las condiciones de crecimiento La cepa de tipo salvaje BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) fue cultivada en medio rico en YEPD (1% extracto de levadura, 2% de peptona, 2% de glucosa). Las células fueron cultivadas a 30 ° C con rotación agitación a 200 rpm en frascos de Erlenmeyer en un &quot;frasco de volumen / volumen medio de&quot; proporción de 5:1. El aislamiento de la fracción mitocondrial crudo <ol><li> Crecer 1 L de la cultura de la cepa de tipo salvaje BY4742 durante 48 h. </li><li> Vierta la cultura en 500 ml de tubos de centrifugación Nalgene. Equilibrar la carga de la cosecha y las células por centrifugación durante 5 minutos a 3000 xg a temperatura ambiente. </li><li> Decantar células sedimentadas sobrenadante y resuspender en 250 ml de dH2O. Pellet células por centrifugación durante 5 minutos a 3000 xg a temperatura ambiente. </li><li> Decantar células sedimentadas sobrenadante y resuspender en 250 ml de dH2O. Pellet células por centrifugación durante 5 minutos a 3000 xg a temperatura ambiente. </li><li> Determinar el peso húmedo del sedimento celular. </li><li> Volver a suspender las células sedimentadas en un tampón de TDT [2 ml de buffer / g (peso húmedo), las células]. </li><li> La transferencia de la suspensión celular a 50 ml tubos de plástico Falcon. </li><li> Gire los tubos a 70 rpm en un agitador durante 20 min a 30 ° C. </li><li> Las células de la cosecha por centrifugación durante 5 minutos a 3000 xg a temperatura ambiente. </li><li> Volver a suspender las células sedimentadas en tampón Zymolyase sin Zymolyase [7 ml de tampón / g (peso húmedo), las células]. </li><li> Pellet células por centrifugación durante 5 minutos a 3000 xg a temperatura ambiente. </li><li> Volver a suspender las células sedimentadas en tampón Zymolyase sin Zymolyase [7 ml de tampón / g (peso húmedo), las células]. </li><li> Transferencia de suspensión celular a un frasco de vidrio. </li><li> Añadir el polvo de Zymolyase-100T [1 mg de Zymolyase-100T / g (peso húmedo), las células] a la suspensión celular. </li><li> Gire el frasco con la suspensión de células a 70 rpm en un agitador durante 30 min a 30 ° C. </li><li> Esferoplastos transferencia formado debido a la digestión de la pared celular con Zymolyase-100T de 50 ml tubos plásticos de centrífuga. </li><li> Pellet esferoplastos por centrifugación durante 8 minutos a 2200 xg a 4 ° C. </li></ol> ** Todos los pasos posteriores deben llevarse a cabo a 4 ° C o en hielo. Las suspensiones de esferoplastos deberá ser manejado suavemente con pipetas con las puntas cortadas para evitar romper orgánulos .** <ol start="18"><li> Resuspender esferoplastos pellets en tampón de homogeneización helada [6,5 ml de buffer / g (peso húmedo), las células]. </li><li> Pellet esferoplastos por centrifugación durante 8 minutos a 2200 xg a 4 ° C. </li><li> Resuspender esferoplastos pellets en tampón de homogeneización helada [6,5 ml de buffer / g (peso húmedo), las células]. </li><li> La transferencia de células a un pre-refrigerados en hielo de vidrio homogeneizador. </li><li> El uso de un mortero apretado, homogeneizar las células, haciendo 15 golpes de la mano del mortero. </li><li> Añadir 1 volumen de tampón de homogeneización helada. </li><li> Transferencia de esferoplastos homogeneizada a 50 ml tubos plásticos de centrífuga. </li><li> Pellet células sin romper, los núcleos y los desechos grandes por centrifugación durante 5 minutos a 1500 xg a 4 ° C. </li><li> Centrifugar el sobrenadante resultante durante 5 minutos a 3000 xg a 4 ° C. </li><li> Centrifugar el sobrenadante resultante durante 15 minutos a 12.000 xga 4 ° C. </li><li> Decantar el sobrenadante y el precipitado se resuspende en helado de tampón de homogeneización [6,5 ml de buffer / g (peso húmedo), las células]. </li><li> Centrifugar durante 5 minutos a 3000 xg a 4 ° C. </li><li> Centrifugar el sobrenadante resultante durante 15 minutos a 12.000 xga 4 ° C. </li><li> Decantar el sobrenadante y el precipitado se resuspende en 3 ml de tampón helado SEM. </li></ol> ** A pesar de esta suspensión se enriquece en la mitocondria, que también contiene otros orgánulos como el retículo endoplasmático (microsomas), aparato de Golgi y vacuolas. Para obtener puro mitocondrias, esta fracción mitocondrial crudo puede ser objeto de fraccionamiento adicional, tal como se describe a continuación .** La purificación de las mitocondrias desprovistas de contaminación por otros orgánulos <ol><li> Coloque 1,5 ml de helado de 60% (w / v) de sacarosa en tampón de EM en un tubo de centrífuga Beckman ultra claro. </li><li> Superposición del 60% (w / v) de sacarosa con 4 ml de 32%, 1,5 ml de 23%, 1,5 ml de 15% de sacarosa (todos buffer p / v en EM). </li><li> Colocar 3 ml de la fracción mitocondrial crudo en el SEM de amortiguación en la parte superior del 15% (w / v) de sacarosa. </li><li> Centrífuga en un Beckman SW41 Ti pivotar-Rotor durante 1 hora a 134.000 xg (33.000 rpm) a 4 ° C. </li><li> La forma de la mitocondria intacta una banda de color marrón en el 60% / interfaz de sacarosa del 32%. </li><li> Retire con cuidado sacarosa hasta llegar a la banda mitocondrial. </li><li> Retire la banda mitocondrial utilizando una pipeta con una punta corta y colóquela en un tubo de centrífuga Beckman de la MLS-5 rotor. </li><li> Llenar el tubo con tampón SEM. </li><li> Pellet de las mitocondrias puro por centrifugación en un rotor MLS-5 durante 30 minutos a 10.000 xg (31.000 rpm) durante 30 minutos a 4 ° C. </li><li> Sobrenadante se decanta conuna pipeta. </li><li> Utilice el puro mitocondrias para el análisis de las funciones mitocondriales, y proteoma mitocondrial lipidome, y el ADN mitocondrial. </li></ol> Reactivos <ol><li> TDT buffer [100 mM Tris/H2SO4 (pH 9,4), 10 mM ditiotreitol] (por 15 ml) <ul style="list-style-type:none;"><li> 1,5 ml de 1 M de tampón Tris/H2SO4 (pH 9,4) </li><li> 150 ml de 1 M de TDT </li><li> Volumen de 15 ml </li></ul></li><li> Buffer Zymolyase [20 mM fosfato de potasio (pH 7,4), 1,2 M sorbitol] (por 100 ml) <ul style="list-style-type:none;"><li> 2 ml de una solución amortiguadora de fosfato de potasio M (pH 7,4) </li><li> 60 ml de 2 M de sorbitol </li><li> Volumen de 100 ml </li></ul></li><li> Tampón de homogeneización [10 mM Tris / HCl (pH 7,4), 0,6 M sorbitol, 1 mM EDTA, 0,2% (w / v) BSA] (por 250 ml] <ul style="list-style-type:none;"><li> 2,5 ml de 1 M Tris / HCl buffer (pH 7,4) </li><li> 75 ml de 2 M de sorbitol </li><li> 500 l de 500 mM EDTA </li><li> 0,5 g de BSA </li><li> Volumen de 250 ml </li></ul></li><li> SEM buffer [10 mM MOPS / KOH (pH 7,2), 250 mM de sacarosa, 1 mM EDTA] (por 250 ml) <ul style="list-style-type:none;"><li> 2,5 ml de 1 M MOPS / KOH (pH 7.2) </li><li> 31,25 ml de 2 M de sacarosa </li><li> 500 l de 500 mM EDTA </li><li> Volumen de 250 ml </li></ul></li><li> EM buffer [10 mM MOPS / KOH (pH 7,2), 1 mM EDTA] (por 250 ml) <ul style="list-style-type:none;"><li> 2,5 ml de 1 M MOPS / KOH (pH 7.2) </li><li> 500 l de 500 mM EDTA </li><li> Volumen de 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 el aislamiento de alto rendimiento de puro mitocondrias de células de levadura. Las mitocondrias purificadas por este método son esencialmente libres de contaminación por otros orgánulos y mantener su integridad estructural y funcional después de su purificación. El método descrito mitocondrias rendimientos que son adecuados para células libres de la reconstitución de los procesos esenciales mitocondrial. Estas mitocondrias de alta pureza también se puede utilizar para el análisis de la e...

purification of mitochondria from yeast cells

Las mitocondrias son el sitio principal de producción de ATP durante el metabolismo aeróbico en eucariotas no fotosintéticos células<sup&gt; 1</sup&gt;. Estos orgánulos complejos también juegan un papel esencial en la muerte celular por apoptosis<sup&gt; 2</sup&gt;, La supervivencia celular<sup&gt; 3</sup&gt;, Desarrollo de los mamíferos<sup&gt; 4</sup&gt;, El desarrollo neuronal y la función<sup&gt; 4</sup&gt;, La señalización intracelular<sup&gt; 5</sup&gt;, Y la longevidad de la regulación<sup&gt; 6</sup&gt;. Nuestra comprensión de estos complejos procesos biológicos controlados por las mitocondrias se basa en métodos robustos para la evaluación de su morfología, la proteína y la composición de los lípidos, la integridad de su ADN, y sus numerosas funciones vitales. La levadura en ciernes<em&gt; Saccharomyces cerevisiae</em&gt;, Una genéticamente y bioquímicamente eucariotas unicelulares manipulable con el genoma y el proteoma anotada bien definido, es un valioso modelo para el estudio de los mecanismos moleculares y celulares subyacentes a las funciones biológicas esenciales de las mitocondrias. Para este tipo de estudios, es fundamental contar con las mitocondrias de alta pureza. Aquí se presenta una descripción detallada de un método rápido y eficaz para la purificación de la mitocondria de levadura. Este método permite el aislamiento de alta pureza, las mitocondrias, que son esencialmente libres de contaminación por otros orgánulos y mantener su integridad estructural y funcional después de su purificación. Las mitocondrias purificadas por este método son adecuados para la libre de células de la reconstitución de los procesos esenciales de la mitocondria y se puede utilizar para el análisis de la estructura mitocondrial y funciones, y proteoma mitocondrial lipidome, y el ADN mitocondrial.

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

Purification of Mitochondria from Yeast Cells

Se describe un método rápido y eficaz para la purificación de las mitocondrias de la levadura<em&gt; Saccharomyces cerevisiae</em&gt;. Este método permite el aislamiento de alto rendimiento de puro mitocondrias, que son esencialmente libres de contaminación por otros orgánulos y mantener su integridad estructural y funcional después de su purificación.

Purificación de las mitocondrias de las células de levadura

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 vistas.  Concordia University. Se describe un método rápido y eficaz para la purificación de las mitocondrias de la levadura Saccharomyces cerevisiae. Este método permite el aislamiento de alto rendimiento de puro mitocondrias, que son esencialmente libres de contaminación por otros orgánulos y mantener su integridad estructural y funcional después de su purificación.

Video: 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.

Aislamiento de células T CD4 + de los ganglios linfáticos del ratón utilizando Miltenyi Purificación 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 ...

Investigación

Biología

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.

Tinción de las proteínas en geles con Coomassie G-250, sin ácido disolvente orgánico y el acético

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.

Purificación de los progenitores del 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.

Purificación por cromatografía de ribosomas de levadura de gran actividad

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.

Imagen confocal de Soltero mitocondriales superóxido Parpadea en corazón intacto o<em&gt; En 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.

Aislamiento de ARNm asociados a la mitocondria de levadura para estudiar los mecanismos de traducción localizada

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.

Aislamiento rápido y purificación de mitocondrias para el trasplante por disociación de tejidos y filtración 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.

Aislamiento y análisis funcional de las mitocondrias de las células cultivadas y ratón de Tejidos

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.

La purificación de todo el genoma de ADN circular Extracromosómica a partir de células eucariotas

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

Purification of Platelets from Mouse Blood

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), and plasma proteins. We describe here purification of platelets from mouse blood using three-fold more iohexol gradient medium relative to blood sample volume and centrifugation in a swinging bucket rotor at 400 x g for 20 min at 20 &#176;C. The recovery/yield of the purified platelets were 18.2-38.5%, and the purified platelets were in a resting state, which did not contain any significant number of RBC and WBC. The purified platelets treated with thrombin showed up to 93% activation, indicating their viability. We confirmed that the purified platelets are sufficiently pure using flow cytometric and microscopic evaluation. These platelets can be used for gene expression, activation, granule release, aggregation, and adhesion assays. This method can be used for purification of platelets from the blood of other species as well.

Here, mouse blood was collected in the presence of an anti-coagulant. The platelets were purified by iohexol gradient medium using low speed centrifugation. The platelets were activated with thrombin to investigate if they were viable. The quality of the purified platelets was analyzed by flow cytometry and microscopy.

Purificación de plaquetas de la sangre del ratón

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), ...

Methods Article

Purificación de las mitocondrias de las células de levadura