This work was supported by grants from the CIHR and the NSERC of Canada. V.I.T. is a CIHR New Investigator and Concordia University Research Chair in Genomics, Cell Biology and Aging.

Materials and methods
Yeast strains and growth conditions
The wild-type strain BY4742 (MAT&alpha; his3&Delta;1 leu2&Delta;0 lys2&Delta;0 ura3&Delta;0) was grown in rich YEPD medium (1% yeast extract, 2% peptone, 2% glucose). Cells were cultured at 30&deg;C with rotational shaking at 200 rpm in Erlenmeyer flasks at a "flask volume/medium volume" ratio of 5:1.
Isolation of crude mitochondrial fraction
<ol>
<li>Grow 1 L of the wild-type strain BY4742 culture for 48 h.</li>
<li>Pour culture in 500-ml Nalgene centrifuge tubes. Balance the load and harvest cells by centrifugation for 5 min at 3000 x g at room temperature.</li>
<li>Decant supernatant and resuspend pelleted cells in 250 ml of dH2O. Pellet cells by centrifugation for 5 min at 3000 x g at room temperature.</li>
<li>Decant supernatant and resuspend pelleted cells in 250 ml of dH2O. Pellet cells by centrifugation for 5 min at 3000 x g at room temperature.</li>
<li>Determine wet weight of cell pellet.</li>
<li>Resuspend pelleted cells in DTT buffer [2 ml of buffer/g (wet weight) cells].</li>
<li>Transfer the cell suspension to 50-ml Falcon plastic tubes.</li>
<li>Rotate the tubes at 70 rpm in a shaker for 20 min at 30&deg;C. </li>
<li>Harvest cells by centrifugation for 5 min at 3000 x g at room temperature.</li>
<li>Resuspend pelleted cells in Zymolyase buffer without Zymolyase [7 ml of buffer/g (wet weight) cells].</li>
<li>Pellet cells by centrifugation for 5 min at 3000 x g at room temperature. </li>
<li>Resuspend pelleted cells in Zymolyase buffer without Zymolyase [7 ml of buffer/g (wet weight) cells].</li>
<li>Transfer cell suspension to a glass flask. </li>
<li>Add the powder of Zymolyase-100T [1 mg of Zymolyase-100T/ g (wet weight) cells] to the cell suspension.</li>
<li>Rotate the flask with cell suspension at 70 rpm in a shaker for 30 min at 30&deg;C. </li>
<li>Transfer spheroplasts formed due to the digestion of the cell wall with Zymolyase-100T to 50-ml plastic centrifuge tubes.</li>
<li>Pellet spheroplasts by centrifugation for 8 min at 2200 x g at 4&deg;C.</li>
</ol>
**All subsequent steps should be carried out at 4&deg;C or on ice. Suspensions of spheroplasts should be handled gently using pipettes with cut tips to avoid breaking organelles.**
<ol start="18">
<li>Resuspend pelleted spheroplasts in ice-cold homogenization buffer [6.5 ml of buffer/g (wet weight) cells].</li>
<li>Pellet spheroplasts by centrifugation for 8 min at 2200 x g at 4&deg;C.</li>
<li>Resuspend pelleted spheroplasts in ice-cold homogenization buffer [6.5 ml of buffer/g (wet weight) cells].</li>
<li>Transfer cells to a pre-chilled on ice glass homogenizer.</li>
<li>Using a tight pestle, homogenize the cells by making 15 strokes of the pestle.</li>
<li>Add 1 volume of ice-cold homogenization buffer.</li>
<li>Transfer homogenized spheroplasts to 50-ml plastic centrifuge tubes. </li>
<li>Pellet unbroken cells, nuclei, and large debris by centrifuging for 5 min at 1500 x g at 4&deg;C.</li>
<li>Centrifuge the resulting supernatant for 5 min at 3000 x g at 4&deg;C.</li>
<li>Centrifuge the resulting supernatant for 15 min at 12000 x g at 4&deg;C.</li>
<li>Decant supernatant and resuspend pellet in ice-cold homogenization buffer [6.5 ml of buffer/g (wet weight) cells].</li>
<li>Centrifuge for 5 min at 3000 x g at 4&deg;C.</li>
<li>Centrifuge the resulting supernatant for 15 min at 12000 x g at 4&deg;C.</li>
<li>Decant supernatant and resuspend pellet in 3 ml of ice-cold SEM buffer.</li>
</ol>
**Although this suspension is enriched in mitochondria, it also contains other organelles such as the endoplasmic reticulum (microsomes), Golgi, and vacuoles. To get pure mitochondria, this crude mitochondrial fraction can be subjected to further fractionation, as described below.**
Purification of mitochondria devoid of contamination by other organelles
<ol>
<li>Place 1.5 ml of ice-cold 60% (w/v) sucrose in EM buffer into a Beckman Ultra-Clear centrifuge tube.</li>
<li>Overlay 60% (w/v) sucrose with 4 ml of 32%, 1.5 ml of 23%, 1.5 ml of 15% sucrose (all wt/v in EM buffer).</li>
<li>Place 3 ml of the crude mitochondrial fraction in SEM buffer on top of 15% (w/v) sucrose. </li>
<li>Centrifuge in a Beckman SW41 Ti swinging-bucket rotor for 1 h at 134000 x g (33000 rpm) at 4&deg;C.&nbsp; </li>
<li>The intact mitochondria form a brown band at the 60%/32% sucrose interface. </li>
<li>Carefully remove sucrose until reaching the mitochondrial band. </li>
<li>Remove the mitochondrial band using a pipette with a cut tip and place it into a Beckman centrifuge tube for an MLS-5 rotor.</li>
<li>Fill the tube with SEM buffer.</li>
<li>Pellet the pure mitochondria by centrifugation in an MLS-5 rotor for 30 min at 10000 x g (31000 rpm) for 30 minutes at 4&deg;C.</li>
<li>Decant supernatant using a pipette.</li>
<li>Use the pure mitochondria for the analysis of mitochondrial functions, mitochondrial proteome and lipidome, and mitochondrial DNA.</li>
</ol>
Reagents
<ol>
<li>DTT buffer [100 mM Tris/H2SO4 (pH 9.4), 10 mM dithiothreitol] (for 15 ml)
<ul style="list-style-type:none;">
<li>1.5 ml of 1 M Tris/H2SO4 buffer (pH 9.4)</li>
<li>150 &mu;l of 1 M DTT</li>
<li>Volume to 15 ml</li>
</ul>
</li>
<li>Zymolyase buffer [20 mM potassium phosphate (pH 7.4), 1.2 M sorbitol] (for 100 ml)
<ul style="list-style-type:none;">
<li>2 ml of 1 M potassium phosphate buffer (pH 7.4)</li>
<li>60 ml of 2 M sorbitol</li>
<li>Volume to 100 ml</li>
</ul>
</li>
<li>Homogenization buffer [10 mM Tris/HCl (pH 7.4), 0.6 M sorbitol, 1 mM EDTA, 0.2% (w/v) BSA] (for 250 ml]
<ul style="list-style-type:none;">
<li>2.5 ml of 1 M Tris/HCl buffer (pH 7.4)</li>
<li>75 ml of 2 M sorbitol</li>
<li>500 &mu;l of 500 mM EDTA</li>
<li>0.5 g of BSA</li>
<li>Volume to 250 ml</li>
</ul>
</li>
<li>SEM buffer [10 mM MOPS/KOH (pH 7.2), 250 mM sucrose, 1 mM EDTA] (for 250 ml)
<ul style="list-style-type:none;">
<li>2.5 ml of 1 M MOPS/KOH buffer (pH 7.2)</li>
<li>31.25 ml of 2 M sucrose</li>
<li>500 &mu;l of 500 mM EDTA</li>
<li>Volume to 250 ml</li>
</ul>
</li>
<li>EM buffer [10 mM MOPS/KOH (pH 7.2), 1 mM EDTA] (for 250 ml)
<ul style="list-style-type:none;">
<li>2.5 ml of 1 M MOPS/KOH buffer (pH 7.2)</li>
<li>500 &mu;l of 500 mM EDTA</li>
<li>Volume to 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.

This method enables the high-yield isolation of pure mitochondria from yeast cells. Mitochondria purified by this method are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. The described method yields mitochondria that are suitable for cell-free reconstitution of essential mitochondrial processes. These highly pure mitochondria can also be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome...

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.

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

Purification of Mitochondria from Yeast Cells

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.

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 Aufrufe.  Concordia University. Wir beschreiben eine schnelle und effektive Methode zur Aufreinigung von Mitochondrien aus der Hefe Saccharomyces cerevisiae. Dieses Verfahren ermöglicht die High-Yield-Isolierung reiner Mitochondrien, die im Wesentlichen frei von Verunreinigungen durch andere Organellen sind und behalten ihre strukturelle und funktionelle Integrität nach ihrer Reinigung.

Serie: 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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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