Bu çalışma, Ulusal Yaşlanma Enstitüsü (AG04418), İlaç Suistimali Ulusal Enstitüsü, Sağlık-Karolinska Enstitüsünde Lisansüstü Ortaklıkları Programı Ulusal Enstitüsü, Karolinska Enstitüsünde, İsveç Araştırma Konseyi, İsveç Beyin Gücü ve İsveç Beyin Vakfı tarafından desteklenmiştir. Birçok yaratıcı destek için sırasıyla Şekil 1 ve 2, Mattias Karlen ve Dr. Giuseppe Coppotelli sayesinde, teknik yardım için Pernold Karin ve Dr. Barry J. Hoffer, çok yararlı öneriler ve tartışma Lars Olson ve Nils-Göran Larsson.

1. Cryosectioning için doku hazırlanması <ol><li> Mevcut etik izni ile uygun olarak, servikal dislokasyon ya da baş kesme ya da hayvan kurban. </li><li> Ilgi hızla dokuları (örn. Beyin) toplamak ve kuru buz (dokuların en iyi morfoloji elde etmek için sıvı nitrojen ile soğutulmuş isopentane veya propan donma gerekebilir) hızla dondurmak . -80 Alüminyum folyo Mağaza dokuların bölümüne ° C &#39;ye kadar hazır. </li><li> Cryosectioning hazırlık dondurulmuş doku gömme. </li><li> -21 14 mikron Kriyostat bölümleri toplayın ° C (sıcaklığını ayarlamak gerekebilir ± 1-2 ° C). Kullanıma hazır olana kadar -20 ° C&#39;de coverslipping olmadan ısıtma bloğu, ve mağaza slaytlar kullanarak Slaytlarınıza çözülme bölümleri. </li></ol> 2. COX histokimya <ol><li> Slaytlar, oda sıcaklığında 1 saat süreyle kurumaya bırakın. Islak filtre kağıdı ile bir slayt boyama odasında slaytlar koyun, şeritler halinde kesilir. Consisten almak içinHer deneme t sonuçlar, zaman gecikmeleri en aza indirmek için en fazla on slaytlar Deneme başına işlemek için tavsiye edilir. </li><li> Kimyasal bir kaput 1X DAB altında hazırlanın, 100 mcM 0.1 M PBS pH = 7.0 c sitokrom. Hızlı Vortex. </li><li> Katalaz sığır 2 mg (2 mg ml -1 veya yaklaşık 4 IU ml -1) ekleyin. Katalaz tüm tahıllar break up vorteks ile iyice karıştırın. </li><li> Uygula 150 - 200 mcL inkübasyon orta her slayt tüm bölümler üzerine eşit şekilde yaymak için pipet kullanın. </li><li> 37 slaytlar 40 dakika inkübe ° C </li><li> Slaytları fazla çözüm çıkarın. Yıkama 0.1 M PBS pH = 7.0 4 kez, her seferinde 10 dakika, slaytlar. </li><li> Islak kağıt şeritler ile slayt boyama odasına slaytlar dönün. </li></ol> 3. SDH histokimya <ol><li> Bir kimyasal kaputu altında 1.5 mM NBT, 130 mM sodyum süksinat, 0.2 mM PMS, 1.0, 0.1 M PBS pH = 7.0 mM sodyum azid hazırlayın. Take korumak için dikkatli olunIşıktan PMS. Hızlı Vortex. </li><li> Her bir slayt için inkübasyon orta 150-200 mcL uygulayın, tüm bölümleri üzerine eşit olarak yaymak için pipet kullanın. </li><li> 37 slaytlar 40 dakika inkübe ° C </li><li> Slaytları fazla çözüm çıkarın. Yıkama 0.1 M PBS pH = 7.0 4 kez, her seferinde 10 dakika, slaytlar. </li><li> % 70,% 70,% 95,% 95,% 99.5: etanol aşağıdaki konsantrasyonları 2 dakika için slaytlar dehydrate. Ardından% 99,5 oranında ek bir adımda 10 dakika izin verir. </li><li> Place 10 dakika için ksilen slaytlar. Entellan ve kapak ile monte edin. Slaytlar havalandırılan bir alanda bir gecede, ya da en azından 1-2 saat kurumasını bekleyin. </li></ol> 4. Mitokondriyal disfonksiyon Belirlenmesi <ol><li> Mitokondriyal disfonksiyon miktarı, hücresel mavi boyanma miktarı ile gösterilir. Bu miktarlar yarı-ölçmek için, slaytlar kodlu ve parlak alan mikroskobu altında görüntülendi olmalıdır. Yarı kantifikasyon kör bir yapılmalıdır<html> temelinde bir ölçek kullanılarak, örneğin 0-4 (0 mavi boyama, 4, sadece mavi boyama). Bu her konu / hayvan için ortalama değerini hesaplamak için belirli bir konuda / hayvan çeşitli bölümler bu tür yarı-kantifikasyon gerçekleştirmek için en iyisidir. </li><li> İstatistik non-parametrik Mann-Whitney veya Kruskal-Wallis testi kullanılarak yapılmalıdır. </li></ol> 5. Uygun özgüllük kontrolleri <ol><li> COX aktivitesi için özgüllük kontroller için, &quot;COX histokimya&quot; adımları yineleyin ve 2.5 mM sodyum azid, bir terminal solunum zinciri inhibitörü ekleyin. </li><li> SDH aktivite için özgüllük kontroller için, ortadan kaldırılması ve 50 mM malonat, SDH rekabetçi bir inhibitörü ilavesi ile sodyum süksinat &quot;SDH histokimya&quot; adımları tekrarlayın. </li><li> Yıkayın ve bir etanol dizi bölümleri kurutmak ve sonra adımları 3.4 &#39;de açıklandığı gibi slaytların montajı ve kapak - 3.6. </li></ol> 6. Temsilcisi Sonuçlar: çadır &quot;&gt; yabani tip ve erken yaşlanma mtDNA Mutator farelerin beyin bölümlerinde uygun COX / SDH çift etiketleme histokimya COX / SDH çift etiketleme histokimyasal assay Şekil 2&#39;de gösterilmiştir. Temsilcisi örnekler genel şeması Şekil gösterilmiştir 3. koyu kahverengi lekelere (Şekil 3, sol panel) wild-tip farelerde, mavi boyama belirtilen solunum zinciri eksiklikleri, hücreler normal COX faaliyet gösterdi 12 haftalık mtDNA Mutator fareler ortaya çıkarıldı ve bu eksiklikler oldu 46 hafta (Şekil 3, orta ve sağ panel) yaş mtDNA Mutator fareler gibi daha yaygın. Yetersiz COX etiketleme nedeniyle wild-tip farelerden alınan beyin bölümlerinde Uygunsuz COX / SDH çift etiketleme örnekleri Şekil 4&#39;te gösterilmiştir. Yetersiz inkübasyon gösteri COX aktivite için zaman, ya da moleküler oksijen inkübasyon sırasında slayt coverslipping durumu azaltarak, DAB tepki azaltılmış birikimi ile sonuçlandı.böylece iyon ürün ve SDH inkübasyon sırasında mavi formazan son ürün oluşumu için izin verdi. (Sol Şekil 5 ve merkez) COX ve SDH faaliyetleri de ayrı ayrı araştırılmalıdır olabilir, ancak sıralı etiketleme SDH inkübasyon sırasında mavi çökelti oluşumu (Şekil 3, orta ve COX eksiklikler nedeniyle hücreleri, tanımlamada yardımcı olur sağda). COX ve SDH faaliyetleri için Özgüllük kontrolleri de yapılabilir (Şekil 5, sağ). <img alt="Şekil 1" src="/files/ftp_upload/3266/3266fig1.jpg" /> Şekil 1 Mitokondriyal solunum Kompleksleri IV. Mitokondriyal solunum zinciri iç zarı içinde bulunan ve beş kompleksleri içerir. Solunum zincirinin amacı, IV ve ATP üretmek için Kompleksi V (ATPaz) tarafından kullanılan iç zarından bir proton degrade oluşturur Bunu yaparken Kompleksi elektron taşıma. Kırmızı altıgenler huknt altbirimden mtDNA tarafından kodlanan. Beyaz altıgenler nükleer DNA (Kompleksi II nükleer genomu tamamen kodlanmış olduğunu unutmayın) tarafından kodlanan alt birimden temsil eder. Bu nedenle, mitokondriyal genom mutasyonları solunum zinciri kompleksleri altbirimden mutasyonlar nedeniyle solunum zinciri disfonksiyona neden olabilir. <img alt="Şekil 2" src="/files/ftp_upload/3266/3266fig2.jpg" /> Şekil 2. COX / SDH çift etiketleme histokimyasal testinin Akış çizelgesi. Ilgi organlarını teşrih, kuru buz dokuların hızla dondurmak ve saklayın - 80 ° C Kriyostat bölümleri toplayın ve tutmak - 20 ° C kadar kullanımda. 1 saat süreyle oda sıcaklığında kurumaya bölümleri izin ver. COX histokimya inkübasyon ortamı hazırlayın, slaytlara uygulamak, 37, 40 dakika boyunca inkübe ° C PBS 4 kez bölümlerde her bir yıkama için 10 dakika yıkayın. SDH histokimya inkübasyon ortamı hazırlayın, sli uygulamakdes ve yine 37 ° C&#39;de 40 dakika inkübe PBS içinde bölümleri tekrar yıkayın, bir etanol serisi kurutmak ve ardından montaj ve slaytlar lamel. COX / SDH çift işaretli bölümleri 1-2 saat içinde aydınlık alan mikroskobu altında görüntülemek için hazır. <img alt="Şekil 3" src="/files/ftp_upload/3266/3266fig3.jpg" /> Şekil 3. COX / SDH çift etiketleme Temsilcisi örnekler. Yabani tip ve erken yaşlanma mtDNA Mutator farelerin beyin bölümleri sırayla COX ve SDH faaliyetleri için etiketli. (Ölçek çubuğu: 200 mikron) Normal COX aktivitesi (koyu kahverengi renk ile gösterilir), wild-tip farelerden (solda) hippocampus bölgesinde görülmüştür. COX eksiklikleri (mavi renkle gösterilir) mtDNA Mutator fareler (merkez sağ) hippocampus bölgesinde ortaya çıkarılmıştır. MtDNA Mutator farelerde yaşı 46 hafta COX aktivitesi daha fazla azalma, solunum zinciri disfonksiyon yaygın alevlenme düşündüren vardı. Gözlenen mitomtDNA Mutator fareler 12 chondrial disfonksiyon mtDNA nokta mutasyonlarının yanı sıra lineer silmeler 5 düzeylerinde artış yüksek düzeyde neden olur. <img alt="Şekil 4" src="/files/ftp_upload/3266/3266fig4.jpg" /> Şekil 4 Uygunsuz COX / SDH çift etiketleme örnekleri. Wild-tip farelerden alınan Beyin bölümleri sırayla COX ve SDH faaliyetleri için etiketli. (Ölçek çubuğu: 200 mikron) COX faaliyet gösterilmesi için yetersiz inkübasyon sürelerinde (10 ve 25 dakika) 40 dakikalık bir inkübasyon süresi (solda ve ortada) ile karşılaştırıldığında, kahverengi DAB reaksiyon ürünü daha düşük bir birikimi sonuçlandı. SDH inkübasyon sırasında mavi formazan son ürün oluşumu için izin kısaltılmış inkübasyon sürelerinde, yanıltıcı COX eksiklikleri bulunan hücrelerin varlığını düşündürmektedir. COX inkübasyon sırasında slaytlar Coverslipping DAB tepki yanlış oluşumu ve birikimi ile sonuçlandıiyon ürün (sağda). <img alt="Şekil 5" src="/files/ftp_upload/3266/3266fig5.jpg" /> Şekil 5. Ndividual COX ve SDH etiketleme ve özgüllük kontrolü. Wild-tip farelerden alınan Beyin bölümleri koyu kahverengi renk ve mavi renk sırasıyla (sol ve merkez) tarafından belirtilen COX ve SDH etkinlikler için ayrı ayrı etiketlenir. COX ve SDH faaliyetleri tek tek etiketli olmasına rağmen, ardışık etiketleme mitokondriyal disfonksiyonu olan hücreleri bulma konusunda avantajlı olduğu kanıtlanmıştır. Yabani tip bir fare beyin COX ve SDH faaliyetleri için özgüllük kontrol etiketleme yokluğu (sağ) bir örnek gösterdi. (Ölçek çubuğu: 200 mikron).

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Çıkar çatışması ilan etti.

Kombine COX / SDH histokimyasal yöntemi mitokondriyal disfonksiyonu olan hücrelerin görselleştirme sağlar. Bu teknik, erken çalışmalar 1968 yılına kadar uzanan, birçok hasta 14,19,26,27 mitokondriyal hastalıkların belirlenmesi için &quot;altın standart&quot; dikkate alınarak, popüler olmaya devam etmektedir. Şimdi, mtDNA mutasyonu odaklı yaşlanmaya ve yaşlanma ile ilişkili bozukluklar 12,13,18,20,21,24 araştırmak için sık sık kullanılır. COX / SDH çift etiketleme yönt...

<table border="1"><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Donmuş karbondioksit </td><td> AGA Gaz AB </td><td> blok biçimiyle </td><td></td></tr><tr><td> Isopentane (2 methylbutane) </td><td> Sigma-Aldrich </td><td> 277258 CAS: 78-78-4 </td><td></td></tr><tr><td> Cyrostat gömme çözümü </td><td> Sakura Finetek </td><td> Doku Tek 4583 </td><td></td></tr><tr><td> Kriyostat </td><td> Microm </td><td> Microm Model HM 500M </td><td></td></tr><tr><td> Slaytlar </td><td> Thermo Scientific </td><td> Super Frost Plus Menzel Gläser J1800AMWZ </td><td></td></tr><tr><td> Kapak gözlük Borosilikat cam </td><td> VWR Uluslararası </td><td> 16004-098 </td><td> 24 x 50 mm </td></tr><tr><td> Filtre Kağıdı </td><td> Munktell Filtre AB </td><td> Kalite: 1350 MakaleSayı: 242 001 </td><td> 430 x 430 mm </td></tr><tr><td> 3,3 &#39;-diaminobenzidin tetrahydrochloride (DAB) </td><td> Sigma-Aldrich </td><td> Sigma Sıvı Yüzey Sistemi, D7304 </td><td></td></tr><tr><td> Sitokrom c (Tip III, kalp at) </td><td> Sigma-Aldrich </td><td> C2506 CAS: 9007-43-6 </td><td></td></tr><tr><td> Katalaz Sığır (karaciğer) </td><td> Sigma-Aldrich </td><td> C9322 CAS: 9001-05-2 </td><td></td></tr><tr><td> Nitroblue tetrazolium (NBT) </td><td> Sigma-Aldrich </td><td> N6876 CAS: 298-83-9 </td><td></td></tr><tr><td> Sodyum süksinat </td><td> Sigma-Aldrich </td><td> S2378 CAS: 6106-21-4 </td><td></td></tr><tr><td> Phenazine methosulfate (PMS) </td><td> Sigma-Aldrich </td><td> P9625 CAS: 299-11-6 </td><td> PMS ışığa duyarlı. Işıktan koruyun. </td></tr><tr><td> Sodyum azid </td><td> Sigma-Aldrich </td><td> S8032 CAS: 26628-22-8 </td><td></td></tr><tr&gt; <td> Ksilen </td><td> VWR Uluslararası </td><td> EM-XX0060-4 </td><td></td></tr><tr><td> Entellan </td><td> VWR Uluslararası </td><td> 100503-870 </td><td></td></tr><tr><td> Malonat (Asit malonik) </td><td> Sigma-Aldrich </td><td> M1296 CAS: 141-82-2 </td><td></td></tr></table>

visualization of mitochondrial respiratory function using cytochrome c oxidase / succinate dehydrogenase (cox/sdh) double-labeling histochemistry

Mitokondriyal DNA (mtDNA) kusurlar, önemli bir hastalık nedenidir ve değişiklikler 1,2 yaşlanmaya ve yaşlanma ile ilgili temelini olabilir . Mitokondriyal teorisi yaşlanma, yaşlanma süreci 3, biyoenerjetik homeostazı ve hücresel fonksiyon değiştirebilir mtDNA mutasyonlarının için bir rol göstermektedir . Kanıtların bir servet bu teori 1,4, 5 mtDNA Mutator fare bir örnek destek derlenmiştir, ancak, yaşlanma mtDNA hasarı kesin rolü 6,7 tamamen anlaşılmış değildir. Solunum enzimleri aktivite Gözlem mitokondriyal disfonksiyonu araştırmak için basit bir yaklaşımdır. Kompleks IV veya sitokrom c oksidaz (COX), mitokondriyal fonksiyon için çok önemlidir. COX katalitik alt birimden mtDNA tarafından kodlanan ve karmaşık montaj (Şekil 1) için gereklidir. Böylece, doğru sentezi ve işlevi büyük ölçüde mtDNA bütünlüğünü 2 dayanmaktadır .Konusu olsa da, diğer solunum yolu kompleksleri incelenmiştir Kompleksleri IV ve II histokimyasal inceleme 8,9 için en müsait. Kompleks II veya süksinat dehidrogenaz (SDH), mitokondriyal biyogenezi 10-12 bir artış gösteriyor olabilir rağmen, tamamen nükleer DNA (Şekil 1) tarafından kodlanır ve faaliyet genellikle etkilenen engelli mtDNA değildir. Engelli mtDNA yaşlanma, mitokondriyal hastalıklarda gözlenen ve yaşla ilişkili hastalıklar genellikle, düşük ya da hiç COX aktivitesi 2,12-14 hücrelerin varlığı yol açar . COX ve SDH faaliyetleri ayrı ayrı incelenmiştir olmasına rağmen, 15,16 ardışık çift etiketleme yöntemi, 12,17-21 mitokondriyal disfonksiyon hücrelerin yerini avantajlı olduğu kanıtlanmıştır. Testin en uygun anayasaların çoğu substrat konsantrasyonu, elektron alıcısı / donörler, ara elektron taşıyıcıları, pH etkisi, ve reaksiyon t gibi tespit edilmiştirime 9,22,23. 3,3 &#39;-diaminobenzidin (DAB), etkili ve güvenilir bir elektron donörü 22 . , Kahverengi indamine polimer ürünü işleyen COX hücrelerinde mitokondriyal cristae yerelleştirme ve hücreler 22 doyurabilecek. Bu nedenle, işlevsiz COX ile bu hücrelerin mavi formazan son ürün 9,24 nitroblue tetrazolium (NBT), bir elektron alıcısı azaltılması SDH aktivite görselleştirme için izin DAB ürün doymuş olmaz. Sitokrom c ve sodyum süksinat substratlar kontrolü ve mutant / hastalıklı dokuların 9 arasında endojen düzeylerini normalleştirmek için eklenir. Katalaz peroksidaz aktivitesini 9,22 mümkün kirlenmesine neden olan reaksiyonları önlemek için bir önlem olarak eklenir. Phenazine methosulfate (PMS), bir ara elektron taşıyıcı, son reaksiyon ürünlerinin 9,25 oluşumunu arttırmak için sodyum azid, bir solunum zinciri inhibitörü ile birlikte kullanılır. Bu bilgilendirmek rağmenation, bu yakışık alır basit bir testin sonucu etkileyen bazı kritik detayları, özgüllük kontrolleri ve tekniği gelişmeler yanı sıra, henüz sunulmuş değil.

Watch this Scientific Journal Video about Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry at JoVE.com

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

Sitokrom c oksidaz / sodyum dehidrogenaz (COX / SDH) çift etiketleme yöntemi, doğrudan bir görünüm için taze dondurulmuş doku kesitlerinde mitokondriyal solunum enzim eksiklikleri sağlar. Bu basit bir histokimyasal teknik ve mitokondriyal hastalıklar, yaşlanma, yaşlanma ile ilişkili bozukluklar araştıran yararlıdır.

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 ...

visualization-mitochondrial-respiratory-function-using-cytochrome-c

Research

JoVE Journal

Biology

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

36.5K Görüntüleme.  Karolinska Institutet. Sitokrom c oksidaz / sodyum dehidrogenaz (COX / SDH) çift etiketleme yöntemi, doğrudan bir görünüm için taze dondurulmuş doku kesitlerinde mitokondriyal solunum enzim eksiklikleri sağlar. Bu basit bir histokimyasal teknik ve mitokondriyal hastalıklar, yaşlanma, yaşlanma ile ilişkili bozukluklar araştıran yararlıdır.

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

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 ...

Biyoloji

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

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 ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

Ex vivo</emVasküler Reaktivite incelenmesi için> İzole İskelet mikrodamar Hazırlık

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

Böbrek Hücreleri aşırı eksprese eden protein kinaz C izozimlerinin Mitokondriyal Fonksiyonlar ve Hücre Canlılık Değerlendirilmesi

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Sox10 içinde Kraniyofasiyal Kalkınma Görselleştirme: kaede Transgenik Zebra balığı Hattı Time-lapse konfokal mikroskopi kullanılarak

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

Büyük ölçekli Gen demonte olarak<em&gt; C. elegans</em&gt; Sağlam Kayıp fonksiyon-fenotipleri oluşturmak için DsRNA Besleme Kitaplıklar kullanma

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

Yarı Tüm vücut pletismografisi kullanılarak Farelerde Solunum Fonksiyon Ölçme

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

Yüksek çözünürlüklü Solunum permeabilize ve bozulmamış Hücreleri Mitokondrial fonksiyonu değerlendirmek için

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Mitokondriyal Yapısı ve İşlevi incelenmesi<em&gt; Drosophila</em&gt; Yumurtalıklar

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.