Bu çalışma, MD, Ulusal Sağlık Enstitüsü, hibe NS042197 tarafından desteklenen oldu. Ajk hibe T32GM008719 ve F30NS068006 tarafından desteklenmiştir.

1. Mikroenjeksiyon İğneleri Üretimi <ol><li> Prefabriklerin mikroenjeksiyon iğneler ticari olarak mevcuttur (örn. Eppendorf Femtotips) ve bir microinjections bir sayıda performans ise yararlıdır. Ancak, uzun vadeli yetenekleri microinjecting kurmak isteyenler için alternatif bir ince duvar borosilikat cam kapilerleri ve ticari bir iğne çektirmenin kullanarak laboratuvar mikroenjeksiyon iğne üretmektir. Bu aynı zamanda farklı hücre türleri için yararlı olabilir çeşitlendirilecek iğne şeklini sağlar. </li><li> Narishige PC-10 Mikroenjeksiyon İğne Çektirme, tüm dört farklı ağırlık takın ve 58.0 göreceli ısı ayarı (No.2 ısıtıcı) ile bir adım çekme programı (Adım 1 ayar) kullanabilirsiniz. Çıkan iki iğne benzer bir uzunluk, böylece her kılcal merkezi ısıtma elemanı yerleştirmek için emin olun. </li><li> Birkaç kılcal damarlar (kılcal damarların yaklaşık 2 ilgi her protein için) çekin. </li><li> Bir kapta saklayın iğne iğne ucu zarar vermemek için emin olmak. Mikroenjeksiyon iğneler, köpük ya da Blu-Tack gibi malzemeler tutmak için konteyner kullanılabilir. </li></ol> 2. Enjeksiyon için protein karışımlar hazırlanması <ol><li> 7.4 pH 1 M potasyum klorür ve 0.1 M potasyum fosfat (KPI) tamponu (K 2 HPO 4 ve KH 2 PO 4 ekimolar karışımı) içeren bir 10x mikroenjeksiyon tampon hazırlayın. Bu tampon, uzun süreli oda sıcaklığında saklanabilir. </li><li> Enjeksiyon görselleştirmek için rodamin-dekstran gibi bir floresan boya eklenmesi gerekiyor. The10x mikroenjeksiyon tampon su seyreltilir ve 20-40 mg / ml rodamin dekstran içeren 5x mikroenjeksiyon tampon çözelti yapmak için rodamin-dekstran tozu çözülür. </li><li> Karanlıkta bu çözüm Mağaza, 4 ° C 100&#39;e kadar bireysel protein çözüm hazırlıkları için yeterli olmalıdır. Floresein izotiyosiyanat-dekstran gibi diğer boyalar, ikame edebilir. </li><li> 20 mg / ml &#39;lik bir konsantrasyon, arıtılmış sitokrom c suda çözünür sitokrom c stokları hazırlayın . -80 Saklayınız sitokrom c ° C uzun süreli depolama ve sitokrom c küçük (~ 10 mcL) alikotları saklayarak, donma-çözülme döngüleri önlemek için. </li><li> 1x tampon (100 mM KCl, 10 mM KP son konsantrasyon 10 mg / ml sitokrom c 3 mcL su ve 5 mcL sitokrom c rodamin-dekstran içeren 2 mcL 5x mikroenjeksiyon tampon birleştirerek enjeksiyon için 10 mcL protein karışımı hazırlayın i, 4-8 mg / ml rodamin dekstran). </li></ol> 3. Sitokrom c Sitoplazmik Mikroenjeksiyon <ol><li> Mikroenjeksiyon hemen önce, 4, 10 dakika 16.000 g sitokrom c ve mikroenjeksiyon tampon karışımı santrifüj ° C mikroenjeksiyon iğneler tıkayabilir herhangi bir partikül madde ayrı. </li><li> Santrifüj sırasında, hava basıncı oluşturmak için izin mikroenjektör açmak. </li><li> Hücrelerin mikroskop sahne ortasına bir tabak koyun ve hücrelere odaklanmak. Hücrelerin inkübatör dışında tutulmasını zaman miktarını en aza indirmek için, hücreleri genellikle inkübatör 30 dakika içinde iade edilir. </li><li> Pipet 0.5-1 kılcal künt ucunu protein karışımı üst yüzeyi mcL. Parçacıkları tüpün dibine santrifüj edilmiştir herhangi bir pipetle için dikkatli olun. Bir dakika içinde, protein karışımı kapiler eylem yoluyla iğne ucu dağıtacak. </li><li> Mikromanipülatör ve pozisyon iğne ucu yaklaşık 45 derecelik bir açıyla mikroskop iletilen ışık geçer böylece kılcal sahibine sıkıca iğne takın. </li><li> Doğrudan görüş alanının merkezinde yer alır, böylece iğne ucunun konumunu ayarlayın. Iğne merkezi için, mikroskop mercek aracılığıyla bakarken iğne taşımak için mikromanipülatör kullanın. Iğne gölge görünür olmalıdır. Iğne, onun gölgesi sadece iğne ucu hücreleri üzerinde merkezli olduğunu belirten görüş alanında bir yarısında görülür şekilde ayarlayın. </li><li> Mikroenjektör Sürekli Flow moduna ayarlayın ve çalışma basıncı 20 - 100 hPa. Her iğne, farklı bir çalışma basıncı gerektirebilir, ve çalışma basıncı, büyük olasılıkla sabit bir akış sağlamak için mikroenjeksiyon işlemi sırasında ayarlanması gerekecektir. </li><li> Yukarıda hücreler sadece bir pozisyon için kaba düğmesini kullanarak iğne indirin. Bunu yapmak için mikroskop odak düzlemine hücreleri üzerinde bir konuma yükseltmek. Sonra kaba düğmesini kullanarak hücrelerin doğru iğne, iğne ucu odak kadar düşük olur. </li><li> Re merkezi görüş alanı içinde iğne ucu ve mikroskop objektif değiştirmeden büyütme oranını artırın. </li><li> Yavaş yavaş, ince manipülatör topuzu unti kullanarak iğne düşükl iğne hücrelerin odak düzlemi üzerinde sadece biraz. </li><li> Rodamin kırmızı floresan protein karışımı akışını kontrol edin. Protein karışımı ince, sürekli olarak iğne çıkmadan olmalıdır. İğneler ve iğne tehlikeye yeniden yüklenmiş değiştirilmesi ya da akışı çok güçlü olmalıdır Bazen, kılcal cam ucu kapalı ve iğne akış görülür. Bu durum ortaya çıkarsa, iğne değiştirmek veya iğne ucu hafifçe rüptürü dikkatlice kültür çanak alt düşük. </li><li> Iğne ucu, yaklaşık 45 derecelik bir açıyla bir hücreyi dönük şekilde yerleştirin. Hücre doğru hareket ederken Sonra tek bir düzgün hareket, iğne düşük. Ikinci bir yumuşak hareket ile, hemen hücre çıkarmak için iğne yönü ters. </li><li> Hücre başarıyla enjekte genellikle biraz şişer ve hücre içinde kırmızı floresan rodamin görselleştirme tarafından teyit edilebilir. Bazen, bir hücrenin çekirdeği, görünür olacaktır yanlışlıkla enjekte edilir. </li><li> Mikroskop aşamasına kadar yaklaşık 50-100 hücreleri enjekte edilir ayarlayarak hücre enjekte etmek için devam edin. Mikroskop sahne taşırken, üst hücreleri temizler böylece mikroenjeksiyon iğne yükseltmek için emin olun. </li></ol> 4. Temsilcisi Sonuçlar: Sitokrom c sitoplazmik mikroenjeksiyon apoptozis sırasında, mitokondride serbest taklit eder. Bu nedenle, beklendiği gibi, fibroblastlar sığır sitokrom c (Şekil 1A) sitozolik mikroenjeksiyon üzerine apoptozis hızla geçmesi. Maya sitokrom c caspases 12 aktive aciz olduğundan, yalnız enjeksiyon prosedürü hücre ölümünden sorumlu olmadığından emin olmak için, maya sitokrom c enjeksiyon, önemli bir kontrol olarak hizmet vermektedir . İlginçtir ki, post-mitotik sempatik nöronlar sitozolik sitokrom c (Şekil 1B) 8,13 dikkat çekici dayanıklıdır. Laboratuvar endojen kaspaz inhibitörü XIAP nöronlar 14 kaspaz aktivasyonu önemli bir inhibitörü olduğunu tespit etmiştir. Bu nedenle, sitokrom c enjeksiyonu takiben ölmek nöronlar için, XIAP ilk inaktive haline gelmelidir . Örneğin, sitokrom c xiap içine mikroenjeksiyon - / - sempatik nöronlar, bu hücrelerde kaspaz aktivasyonu ve apoptozis (Şekil 2) için yeterli . <img src="/files/ftp_upload/2773/2773fig1.jpg" alt="Şekil 1"/> Şekil 1. Sitokrom c Sitoplazmik mikroenjeksiyon nöronlar fibroblastlar hızlı ölüme neden olur, ama değil . A) Wild-tip MEFS veya (B) doğum sonrası 5 gün yabani tip sempatik nöronlar ile birlikte enjekte hücreleri işaretlemek için rodamin-dekstran sığır sitokrom c (10 mg / ml) ile microinjected. Görüntü aynı hücreleri hemen sonra enjeksiyon (0 saat) alanında, ya da belirtilen zaman gösterir. Oklar enjekte edilen hücreler gösterir. Ölçeği bar, 20 mm. <img src="/files/ftp_upload/2773/2773fig2.jpg" alt="Şekil 2"/> Şekil 2. XIAP eksikliği nöronların sitoplazmik sitokrom c mikroenjeksiyon duyarlı. XIAP knockout farelerde postnatal 5. günde sempatik nöronlar enjekte hücreleri işaretlemek için rodamin-dekstran ile birlikte sığır sitokrom c (10 mg / ml) ile microinjected. Görüntüler hücreler hemen aşağıdaki enjeksiyon alanında aynı (0 saat), veya 5 saat sonra sitokrom c mikroenjeksiyon (5 saat) göstermektedir. Ölçeği bar, 20 mm.

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Tüm hayvanlar üzerinde deneysel prosedürleri Kuzey Carolina Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi.

Sitokrom c, doğrudan hücre sitoplazma içine mikroenjeksiyon apopitoz sonrası mitokondriyal düzenleme çalışmaları için olanak sağlayan bir benzersiz ve güçlü bir araçtır. Önemlisi, bu teknik, hücresel veya mitokondriyal hasara yol açan ajanların kullanımı olmadan mitokondri apoptozis downstream doğrudan aktivasyonu için olanak sağlar. Bu protokol, apoptozis çalışmaları için sitokrom c mikroenjeksiyon odaklanmış olsa da, genel ilkeleri Burada g...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> DM IRE2 Ters Mikroskop </td><td> Leica </td><td></td><td></td></tr><tr><td> PC-10 Mikroenjeksiyon İğne Çektirme </td><td> Narishige </td><td></td><td></td></tr><tr><td> MWO-202 Mikromanipülatör </td><td> Narishige </td><td></td><td></td></tr><tr><td> FemtoJet mikroenjektör </td><td> Eppendorf </td><td></td><td></td></tr><tr><td> Microfilament İnce duvar Boroscilicate Kılcal Cam </td><td> AM Sistemleri </td><td> 615000 </td><td> 4 inç uzunluğunda, 1.00 mm dış çapı, 0,75 mm iç çapı </td></tr><tr><td> Rodamin B izotiyosiyanat-Dextran </td><td> Sigma-Aldrich </td><td> R9379 </td><td> Ortalama moleküler ağırlık ~ 70.000 Da </td></tr><tr><td> Sığır Sitokrom c Protein </td><td> Sigma-Aldrich </td><td> C3131 </td><td></td></tr></tbody></table>

activation of apoptosis by cytoplasmic microinjection of cytochrome c

Apoptozis veya programlanmış hücre ölümüne yardımcı hücreleri ölür 1 tarafından korunmuş ve oldukça düzenli bir yoludur . Hücrelerin sitotoksik gerilmeler geniş bir yelpazede karşılaştığınızda Apoptoz tetiklenebilir. Bu hakaretler, sonuçta, mitokondriyal arası boşlukta sitoplazma 2 sitokrom c salınımına neden sinyal kaskadlar başlatabilir. Mitokondri sitokrom c salınımı caspases, sonuçta 3-4 hücre ölümü yürütmek en önemli hücresel proteazlar hızlı aktivasyonunu tetikler kilit bir olay. Apoptozis yol mitokondri 5 sitokrom c serbest memba ve mansap noktalarında düzenlenir. Kaspaz aktivasyonu sonrası mitokondriyal yönetmelik çalışması için, birçok araştırmacılar, holocytochrome c (heme bağlı) hücrelerin içine protein 6-9 sitoplazmik doğrudan mikroenjeksiyon çevirdiler . Sitokrom c normalde heme grubu eki, 10-11 apoptozis etkinleştirmek için sağlamak için gerekli olduğu mitokondri lokalize olur . Bu nedenle, doğrudan caspases etkinleştirmek için, sitokrom c cDNA yapıları ifade mitokondriyal hedefleme ve hem eki neden olurken, sitozolik caspases saklamış olacak çünkü holocytochrome c protein yerine cDNA enjekte etmek gereklidir. Böylece, saflaştırılmış heme bağlı sitokrom c protein doğrudan sitozolik mikroenjeksiyon, hücresel ve mitokondriyal hasara yol açan toksik hakaret kullanmadan mitokondrial sitokrom c salınımı ve apoptoz taklit etmek için yararlı bir araçtır. Bu makalede, fare embriyonik fibroblastlar (MEFS) ve örnek olarak birincil sempatik nöronlar kullanarak, sitokrom c proteinin hücre içine mikroenjeksiyon için bir yöntem açıklanmaktadır. Bu protokol apopitoz araştırmalar için sitokrom c enjeksiyon odaklanırken, burada gösterilen teknikleri da kolayca diğer ilgi proteinlerin mikroenjeksiyon için adapte olabilir.

Watch this Scientific Journal Video about Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c at JoVE.com

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Bu protokol, sitokrom doğrudan sitoplazmik mikroenjeksiyon<em&gt; C</em&gt; Fibroblastlar ve birincil sempatik nöronlar içine protein. Bu teknik, sitokrom giriş için izin verir<em&gt; C</em&gt; Hücreleri ve taklit sitokrom serbest sitoplazma içine protein<em&gt; C</em&gt; Mitokondri, apoptozis sırasında oluşur.

Sitokrom Sitoplazmik Mikroenjeksiyon Apoptoz aktivasyonu C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

activation-of-apoptosis-by-cytoplasmic-microinjection-of-cytochrome-c

Research

JoVE Journal

Biology

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

17.3K Görüntüleme.  University of North Carolina. Bu protokol, sitokrom doğrudan sitoplazmik mikroenjeksiyon C Fibroblastlar ve birincil sempatik nöronlar içine protein. Bu teknik, sitokrom giriş için izin verir C Hücreleri ve taklit sitokrom serbest sitoplazma içine protein C Mitokondri, apoptozis sırasında oluşur.

Video: Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques.   In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.  Central to these studies is CD1d - the antigen presenting molecule that presents glycolipids to NKT cells.  The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells.  Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy.  Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmunity, clearance of infection, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.

CD1d glikolipid Antijen Tanıtım ve NKT hücre Etkinleştirme Terapötik Potansiyeli: Röportaj

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious ...

Biyoloji

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Mikroenjeksiyon Kararlı Transgenik C. elegans Üretimi

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

Transgenik, Üretimi C. elegans Biyolistik Dönüşüm

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

Mikroenjeksiyon tarafından Memeli Hücreleri mRNA Nükleer İhracat Kinetik Analizi

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Polarize Epitel Hücreleri içine Plazmidler, Mikroenjeksiyon Küçük GTPases Fonksiyon Analizi

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

Sitokrom kullanarak Mitokondriyal Solunum Fonksiyon Görselleştirme C Oksidaz / Succinat Dehidrogenaz (COX / SDH) Çift-etiketleme Histokimya

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are evident. A means to detect apoptosis in its earliest, reversible stages would afford a pre-clinical 'window' during which preventive or therapeutic measures could be taken to protect the heart from permanent damage. We present herein a simple and robust method to conjugate human Annexin V (ANX), which avidly binds to cells in the earliest, reversible stages of apoptosis, to superparamagnetic iron oxide (SPIO) nanoparticles, which serve as an MRI-detectable contrast agent. The conjugation method begins with an oxidation of the SPIO nanoparticles, which oxidizes carboxyl groups on the polysaccharide shell of SPIO. Purified ANX protein is then added in the setting of a sodium borate solution to facilitate covalent interaction of ANX with SPIO in a reducing buffer. A final reduction step with sodium borohydride is performed to complete the reduction, and then the reaction is quenched. Unconjugated ANX is removed from the mix by microcentrifuge filtration. The size and purity of the ANX-SPIO product is verified by dynamic light scattering (DLS). This method does not require addition to, or modification of, the polysaccharide SPIO shell, as opposed to cross-linked iron oxide particle conjugation methods or biotin-labeled nanoparticles. As a result, this method represents a simple, robust approach that may be extended to conjugation of other proteins of interest.

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes.

Bir sentezi<em&gt; In vivo</em&gt; MR-saptanabilir Apoptoz Probe

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

Gen Yönetmelik Tek molekül Görüntüleme<em&gt; In vivo</em&gt; Dilinim tarafından Cotranslational Aktivasyon (CoTrAC) kullanma

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Aktif Kaspaz 3 Algılama Tüm montaj immünofloresans tarafından Zebra balığı Embriyolar Apaptozun Analizi

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA (dsDNA) which has the requisite characteristics to stimulate the cytoplasmic DNA sensing pathways can be generated cheaply and with ease. By the concatemerization of short, synthetic oligonucleotides (which lack CpG motifs), dsDNA can be generated to be of sufficient length to activate the cytosolic DNA sensing pathway. This protocol involves blunt end ligation of the oligonucleotides in the presence of polyethylene glycol (PEG), which provides an environment for efficient ligation to occur. The dsDNA concatemers can be used, following purification by phenol/chloroform extraction, to simulate the innate immune response in vitro by standard transfection protocols. This DNA can also be used to stimulate innate immunity in vivo by intradermal injection into the ear pinna of a mouse, for example. By standardizing the concatemerization process and the subsequent stimulation protocols, a reliable and reproducible activation of the innate immune system can be produced.

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

The aim of the protocol is to use optimal methods for stimulating the cytoplasmic DNA sensing pathways in cells and in vivo. This is achieved by improving the generation of long, double-stranded DNA during blunt-end ligation. Cells or mice are then transfected using a lipid transfection reagent.

Sitoplazmik DNA Algılama Pathways uyarılması<em&gt; İn Vitro</em&gt; Ve<em&gt; In Vivo</em

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...