Bu çalışma CIHR ve Kanada NSERC hibe ile desteklendi. VIT CIHR Yeni Araştırmacı ve Genomik Concordia Üniversitesi Araştırma Başkanı, Hücre Biyolojisi ve Yaşlanma.

Gereç ve yöntem Maya suşları ve büyüme koşulları Yabani tip suşu BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) (% 1 maya özü,% 2 pepton,% 2 glukoz), zengin bir YEPD ortamda yetişmiştir . Hücreler, 30 ° C dönüş 05:01 &quot;balon hacim / orta hacimli&quot; oranı Erlenmeyer matara 200 rpm&#39;de sallayarak kültüre edildi. Ham mitokondrial fraksiyon izolasyonu <ol><li> 48 saat için 1 L yabani tip suşu BY4742 kültür büyütün </li><li> 500 ml Nalgene santrifüj tüplerine kültür dökün. 3000 xg&#39;de 5 dakika oda sıcaklığında santrifüj yoluyla yük ve hasat hücreleri Dengesi. </li><li> Durusu supernatant ve tekrar süspansiyon pelet hücreleri dH2O 250 ml. 3000 xg&#39;de 5 dakika oda sıcaklığında santrifüj yoluyla Pelet hücreleri. </li><li> Durusu supernatant ve tekrar süspansiyon pelet hücreleri dH2O 250 ml. 3000 xg&#39;de 5 dakika oda sıcaklığında santrifüj yoluyla Pelet hücreleri. </li><li> Hücre pelletini ıslak ağırlık belirleyin. </li><li> DTT tampon [2 ml tampon / g (ıslak ağırlık) hücreleri] pelet hücreleri tekrar </li><li> 50 ml Falcon plastik tüpler hücre süspansiyonu aktarın. </li><li> Tüpler, 30 ° C&#39;de 20 dakika için bir çalkalayıcı 70 rpm&#39;de Döndür </li><li> 3000 xg&#39;de 5 dakika oda sıcaklığında santrifüj Hasat hücreleri. </li><li> Zymolyase [tampon / g (ıslak ağırlık) hücreleri 7 ml] olmadan Zymolyase tampon pelet hücreleri tekrar </li><li> 3000 xg&#39;de 5 dakika oda sıcaklığında santrifüj yoluyla Pelet hücreleri. </li><li> Zymolyase [tampon / g (ıslak ağırlık) hücreleri 7 ml] olmadan Zymolyase tampon pelet hücreleri tekrar </li><li> Hücre süspansiyonu bir cam erlene aktarın. </li><li> Zymolyase-100T tozu ekleyin [1 mg Zymolyase-100T / g (ıslak ağırlık) hücrelerinin] hücre süspansiyonu. </li><li> , 30 ° C&#39;de 30 dakika için bir çalkalayıcı 70 rpm&#39;de hücre süspansiyonu ile şişesi döndürün </li><li> Transferi spheroplasts Zymolyase-100T için 50 ml plastik santrifüj tüplerine ile hücre duvarının sindirim nedeniyle kurdu. </li><li> Pelet 4 2200 xg&#39;de 8 dakika süreyle santrifüj spheroplasts ° C </li></ol> ** Sonraki tüm adımlar, 4 ° C veya buz üzerinde yapılmalıdır. Spheroplasts, Cezalar organelleri kırılma önlemek için kesme uçları ile hafifçe pipetler kullanarak ele alınmalıdır .** <ol start="18"><li> Buz gibi soğuk homojenizasyon tampon tampon / g (ıslak ağırlık) hücreleri 6,5 ml] pelet spheroplasts tekrar </li><li> Pelet 4 2200 xg&#39;de 8 dakika süreyle santrifüj spheroplasts ° C </li><li> Buz gibi soğuk homojenizasyon tampon tampon / g (ıslak ağırlık) hücreleri 6,5 ml] pelet spheroplasts tekrar </li><li> Buz cam homojenizatör önceden soğutulmuş hücreleri aktarın. </li><li> Sıkı bir havaneli kullanarak, havaneli 15 vuruş yaparak hücreleri homojenize. </li><li> Buz homojenizasyon tampon 1 hacim ekleyin. </li><li> 50 ml plastik santrifüj tüplerine homojenize spheroplasts aktarın. </li><li> Pelet 4 1500 xg&#39;de 5 dakika santrifüj kesintisiz hücreleri, çekirdek, büyük bir enkaz ° C </li><li> 4 3000 xg&#39;de 5 dakika süpernatant Santrifüj ° C. </li><li> 4 ° C&#39;de xg 12000 az 15 dakika boyunca ortaya çıkan süpernatant Santrifüj </li><li> Durusu süpernatant ve tekrar süspansiyon pelet buz homojenizasyon tampon tampon / g (ıslak ağırlık) hücreleri 6,5 ml. </li><li> 3000 xg&#39;de 5 dakika santrifüj 4 ° C </li><li> 4 ° C&#39;de xg 12000 az 15 dakika boyunca ortaya çıkan süpernatant Santrifüj </li><li> Durusu süpernatant ve tekrar süspansiyon pelet buz SEM tampon 3 ml. </li></ol> ** Bu süspansiyon mitokondri zenginleştirilmiş olmasına rağmen, aynı zamanda (mikrozomları) endoplazmik retikulum, golgi ve vakuollerde gibi diğer organelleri içerir. Saf mitokondri almak için, bu ham mitokondrial fraksiyon aşağıda açıklandığı gibi, daha fazla fraksiyonasyon tabi olabilir .** Kontaminasyon mitokondri yoksun diğer organellerin saflaştırılması <ol><li> Beckman Ultra Clear santrifüj tüpü içine EM tamponunda% 60 buz koyun 1,5 ml sakkaroz (w / v). </li><li> Yerleşimi% 60 (w / v) 4 ml% 32 ile sakaroz, 1.5 ml% 23,% 15 sakaroz (EM wt / v tampon) 1,5 ml. </li><li> 3 ml,% 15 (w / v) sakaroz üst SEM tampon ham mitokondriyal fraksiyonu yerleştirin. </li><li> 4 134000 xg (33000 rpm) 1 saat ° C Beckman SW41 Ti sallanan kovalı rotor içinde Santrifüj </li><li> % 60 /% 32 sakaroz arayüzü sağlam mitokondri formu kahverengi bir bant. </li><li> Mitokondriyal bant ulaşana kadar dikkatlice sakaroz çıkarın. </li><li> Bir kesim ucu ile bir pipet kullanarak mitokondriyal bandı çıkarın ve bir MLS-5 rotor için bir Beckman santrifüj tüpü içine yerleştirin. </li><li> SEM tamponu ile tüp doldurun. </li><li> Pelet, 4, 30 dakika süreyle 10000 xg (31000 rpm) &#39;de 30 dakika süreyle bir MLS-5 rotor santrifüj saf mitokondri ° C </li><li> Kullanarak Durusu süpernatantbir pipetle. </li><li> Mitokondriyal fonksiyonları, mitokondriyal proteom ve lipidome ve mitokondriyal DNA analizi için saf mitokondri kullanın. </li></ol> Reaktifler <ol><li> DTT tampon [100 mM Tris/H2SO4 (pH 9.4), 10 mM dithiothreitol] (15 ml için) <ul style="list-style-type:none;"><li> 1,5 ml 1 M Tris/H2SO4 tampon (pH 9.4) </li><li> 1 M DTT 150 ul </li><li> 15 ml hacim </li></ul></li><li> Zymolyase tampon [20 mM potasyum fosfat (pH 7.4), sorbitol 1,2 M] (100 ml) <ul style="list-style-type:none;"><li> 2 ml 1 M potasyum fosfat tamponu (pH 7.4) </li><li> Sorbitol 2 M 60 ml </li><li> 100 ml hacim </li></ul></li><li> Homojenizasyon tampon [10 mM Tris / HCl (pH 7.4), 0.6 M sorbitol, 1 mM EDTA,% 0.2 (w / v) BSA] (250 ml.] <ul style="list-style-type:none;"><li> 1 M Tris / HCl tamponu (pH 7.4) 2.5 ml </li><li> Sorbitol 2 M 75 ml </li><li> 500 mM EDTA 500 ul </li><li> BSA 0.5 g </li><li> 250 ml hacim </li></ul></li><li> SEM tampon [10 mM MOPS / KOH (pH 7.2), 250 mM sukroz, 1 mM EDTA] (250 ml) <ul style="list-style-type:none;"><li> 1 M MOPS / KOH tamponu (pH 7.2) 2.5 ml </li><li> 31.25 ml 2 M sakkaroz </li><li> 500 mM EDTA 500 ul </li><li> 250 ml hacim </li></ul></li><li> EM buffer [10 mM MOPS / KOH (pH 7.2), 1 mM EDTA] (250 ml) <ul style="list-style-type:none;"><li> 1 M MOPS / KOH tamponu (pH 7.2) 2.5 ml </li><li> 500 mM EDTA 500 ul </li><li> 250 ml hacim </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.

Bu yöntem, maya hücreleri saf mitokondri yüksek verimli izolasyon sağlar. Bu yöntemi ile saflaştırılmış Mitokondri kontaminasyon diğer organellerin aslında ücretsiz ve kendi arıtma sonrası yapısal ve fonksiyonel bütünlüğünü korumak. Temel mitokondriyal süreçlerin hücre sulandırıldıktan için uygun olan mitokondri açıklanan yöntem verim. Bu son derece saf mitokondri mitokondriyal yapı ve işlevleri, mitokondriyal proteom ve lipidome ve mitokondriyal DNA analizi için de kullanılabilir.

purification of mitochondria from yeast cells

Mitokondri, ATP üretimi ana site ökaryotik olmayan fotosentetik hücrelerde aerobik metabolizma sırasında<sup&gt; 1</sup&gt;. Bu kompleks organellere apoptotik hücre ölümü de önemli rol oynamaktadır<sup&gt; 2</sup&gt; Hücre sağkalım<sup&gt; 3</sup&gt; Memeli gelişme<sup&gt; 4</sup&gt; Nöronal gelişimi ve işlevi<sup&gt; 4</sup&gt;, Hücre içi sinyal<sup&gt; 5</sup&gt; Ve uzun ömürlü düzenleme<sup&gt; 6</sup&gt;. Mitokondri tarafından kontrol edilen bu karmaşık biyolojik süreçlerin anlayışımız, morfoloji, protein ve lipid bileşimi, onların DNA bütünlüğünü ve sayısız hayati fonksiyonlarını değerlendirmek için sağlam yöntemler dayanmaktadır. Maya<em&gt; Saccharomyces cerevisiae</em&gt; Açıklamalı genomu ve iyi tanımlanmış proteom, genetik ve biyokimyasal manipulable tek hücreli ökaryot mitokondri temel biyolojik fonksiyonları altında yatan moleküler ve hücresel mekanizmaları çalışmak için değerli bir model. Bu tür çalışmalar için, son derece saf mitokondri sahip olmak çok önemlidir. Burada mitokondri maya arıtma için hızlı ve etkili bir yöntemin ayrıntılı bir açıklama mevcut. Bu yöntem aslında diğer organeller tarafından kontaminasyon ücretsiz ve kendi arıtma sonrası yapısal ve işlevsel bütünlüğü korumak son derece saf mitokondri izolasyonu sağlar. Bu yöntem ile arındırılmış Mitokondri hücre sulandırıldıktan temel mitokondriyal süreçleri için uygundur ve mitokondriyal yapı ve işlevleri, mitokondriyal proteom ve lipidome ve mitokondriyal DNA analizi için kullanılabilir.

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

Purification of Mitochondria from Yeast Cells

Biz maya mitokondri arıtma için hızlı ve etkili bir yöntem tarif<em&gt; Saccharomyces cerevisiae</em&gt;. Bu yöntem aslında diğer organeller tarafından kontaminasyon ücretsiz ve kendi arıtma sonrası yapısal ve fonksiyonel bütünlüğünü korumak saf mitokondri yüksek verimli izolasyon sağlar.

Maya hücreleri Mitokondri saflaştırılması

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 Görüntüleme.  Concordia University. Biz maya mitokondri arıtma için hızlı ve etkili bir yöntem tarif Saccharomyces cerevisiae. Bu yöntem aslında diğer organeller tarafından kontaminasyon ücretsiz ve kendi arıtma sonrası yapısal ve fonksiyonel bütünlüğünü korumak saf mitokondri yüksek verimli izolasyon sağlar.

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.

CD4 İzolasyon Miltenyi MACS Arıtma Fare lenf düğümleri + T hücreleri

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

Biyoloji

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.

Organik Solvent ve Asetik Asit olmadan Jeller Coomassie G-250 ile Proteinlerin Boyama

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.

İskelet Kası progenitörlerin saflaştırılması

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.

Yüksek Aktif Maya Ribozomlar Kromatografik Arıtma

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.

Tek Mitokondriyal süperoksit Bozulmamış Kalp Flaşlar veya konfokal Görüntüleme<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.

Lokalize Çeviri Mekanizmaları Eğitim için Maya Mitokondri ile mRNAlarının Associated İzolasyonu

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.

Doku Ayrışma Ve Diferansiyel süzülerek Nakli İçin Mitokondri Of Rapid İzolasyon ve saflaştırma

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.

İzolasyon ve Kültür Hücrelerinin ve Fare Doku gelen Mitokondri Fonksiyonel Analizi

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.

Ökaryotik Hücrelerde gelen ekstrakromozomal dairesel DNA genom Arıtma

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.