Yazarlar mRNA çıkarma ve PCR ile yardım için Dr. Paul Witting ve Bayan Genevieve Fong kabul etmek isteyen, ve destek için Garnett Passe ve Rodney Williams Memorial Vakfı.

Etik Beyanı: Aşağıda belirtilen tüm işlemler Sydney Hayvan Etik Kurulu Üniversitesi tarafından onaylanmıştır. 1. Hayvanlar NOT: 1 ve 8 - 9 aylık fareler (C57 / BL6) Hayvan Kaynakları Merkezi (Perth, Avustralya) elde edilmiştir. Fareler Sydney Üniversitesi&#39;nde Bosch Kemirgen Tesisinde yerleştirildiler. <ol><li> Gıda ve su ad libitum erişimi olan bir 12/12 saat ışık / karanlık döngüsünde standart fare kafesleri Ev fareleri. </li><li> Yakın kızılötesi her bir yaş grubunda Böl fareler (NUR) tedavi, ya da sahte karşılaştırma için (kontrol) gruplar muamele. </li></ol> 2. yakın kızılötesi (NIR) Işınlama ve Sham Tedavisi <ol><li> Tedavi rejiminin tamamlanmasından önce saç hızlı yeniden büyümesini önlemeye mümkün olduğunca yakından bir elektrikli ustura ile fare baş ve boyun bölgesinde kürk Tıraş. Farelerin patojensiz durumunu muhafaza etmek için,% 70 etanol kullanımıtıraş hayvanlar ve ayrı kafeslere gelen hayvanlar arasındaki F10 veteriner dezenfektan arasındaki aletleri temizlemek için. Hayvanlar üzerinde saplı değildir bu yüzden tedaviye başlamadan 3 gün önce - bu 2 yapın. </li><li> Kuyruk proksimal ucunu tutarak fare dizginlemek ve yanlış sonuçlara yol açabilir stresi en aza indirecek şekilde o bir el ya da tezgah üstünde avuç içinde dinlenmek için izin verir. </li><li> LED Nir (670 nm) (ışık yayan diyot) 1 Cihazı tutun - 2 cm uzakta maruz (traş) alan ve 90 saniye (Şekil 1A) için cihazı açın. NOT: 90 saniye maruz kalmanın bir sonucu olarak sıcaklık değişimi, 100 ml su içinde 0.2 ° C &lt;olarak ölçülmüştür. </li><li> Hayvanların sahte tedavi grubu için 2.3 ancak aygıt kapalı (Şekil 1B) bırakın - Tekrar 2.2 adımları. </li><li> Hayvanların Nir-bloke tedavi grubu için 2.3 ama alüminyum folyo ile cihazı kapak - yineleyin 2.2 adımları. </li><li> Approximat günlük 2.5 - 2.2 tekrarlayın adımlarıbirbirini takip eden 5 gün süre ile 24 saat aralıklarla, e. </li><li> Beşinci gün tedavi sonrası her iki kulaklarından vestibüler duyu epitel (3 krista ve 1 utricular makülayı) Extract (aşağıdaki Bölüm 3&#39;e bakın). </li></ol> 3. Doku Ekstraksiyon 20 <ol><li> 26 NaHCO 3, 11 glukoz, 250 gliserol, 2.5 KCl, 1.2 NaH 2 PO 4, 1.2 MgCl2, ve 2.5 CaCl2 (mM olarak) oluşan, bir gliserol bazlı yapay serebrospinal akışkan (ACSF) 300 ml hazırlayın. CaCI2 ilave edilmeden önce, 7.4 bir pH değeri ve kalsiyum çökelmesi (bulanıklık) önlemek için karbojen (% 95 O2 ve% 5 CO2) ile çözelti gaz. Bir buz bulamaç oluşturulur, böylece, 45 dakika için -80 ° C dondurucu içinde bir çözüm soğutun. </li><li> Etiketli vida üst mikrotüplerde RNA izolasyonu lizis tamponu hazırlayın (kapak haşhaş durduğunda donma ve sıvı azot içinde çözülme arasındaki boru basınç değişiklikleri) göreüretici veya olağan laboratuar uygulamalarının talimatlarına. Bir alüminyum Dewar şişede sıvı azot hazır bulundurun. NOT: Sıvı azot kullanırken koruyucu kıyafet ve Eyeware kullanın. Doğal gaz formunda hacmi yaklaşık olarak sıvı halde 700 katıdır ve boğulmaya neden olabilir gibi sıvı nitrojen iyi havalandırılmış bir odada kullanılan emin olun. </li><li> Derin bir intra-peritonal enjeksiyon yoluyla ketamin (400 mg / kg) ile fareler uyuşturan. Arka-bacak refleks tamamen fareler tamamen anestezi olduğu göstergesi olarak düşmesi. </li><li> Keskin paslanmaz çelik makas ile fareler başını kesmek ve bir jilet kullanarak kafatasının sagital cilt boyunca bir kesi yapmak (# 22 yuvarlanmış). Bu noktada ve genelinde 3.5 adımları - 3.9, doku üzerinde buz soğuk ACSF düzenli uygulanması ile mümkün olduğunca serin kafatası tonoz, beyin, ve altta yatan vestibüler aparat tutun. </li><li> Bir hızlı s yapmak standart desen makas sivri kolunu kullanmal lambda kafatası insizyon ve sagital sütür boyunca kesilir. </li><li> Yavaşça beyin sürükleyerek olmadan kemik alt yüzeyine mümkün olduğunca yakın bıçak tutarak parietal kemikte altında sığ rongeurs bir kolunu kaydırın. Güvenli ve beyin maruz kadar posteriora yanal parietal kemik ve oksipital kemik uzak çekin. </li><li> Bir küçük paslanmaz çelik spatula kullanın ve vestibülokohlear sinir (CN VIII) maruz uzakta anterior ve orta kranial fossa beyni kaldırın. Doğrudan vestibüler saç hücrelerini innerve primer afferent aksonlar gereksiz gerilimi önlemek için sinir transect. </li><li> CN VIII kesisi sonrasında toto beyin çıkarın. </li><li> Koklea ve orta kranial fossada periferik vestibüler organları içeren kemik labirent gözlemleyin. Her kemik labirent yanında iki küçük kesiler yapmak ve ön semisirküler kanal tutan ve daha sonra çekerek tüm yapıyı tüketimmüttefik. </li><li> Hemen sürekli karbojen ile perfüze ise (aşama 3.1&#39;de tarif edildiği gibi), buz soğukluğunda ACSF çözeltisini ihtiva eden bir kesme çanak kesilerek çıkarılmış labirentler bırakın. </li><li> Steromikroskopla, koklea ile labirenti basılı tutun ve forseps ile çanak altına sabitleyin. <ol><li> Ön semisirküler kanal (SSC) ampulla üstünde kemik küçük bir açıklık kazımak için düz ince forseps kullanın. </li><li> Yavaşça hemen kemik altına ulaşan ve dışa uzak ampulla gelen iterek bu açılış büyütmek. Ağzına forseps itin ve aşağıda kırılgan membranöz labirent zarar değil burada dikkatli. Kesecik, ön ve yan ampülleri tüm maruz kadar bu şekilde devam ediniz. Mümkünse de arka ampulla çıkarın. </li></ol></li><li> Ince forseps kullanarak, hafifçe kaldırın ve kırbacık tamamen müstakil kadar kemik labirent uzak ampülleri. Mümkünse, onları tutmakilişkili semisirküler kanallar tarafından duyusal epitel zarar vermemek için. Bazı durumlarda, yarım daire membranöz kanalın proksimal kısmı kemikten ampülleri serbest bırakmak için iris makas ile kesilebilir gerekebilir. </li><li> Güvenli adımda 3.2 önceki hazırlanan lizis tamponu içine forseps ucu ile yer arasındaki vestibüler organları kavramak. Yavaşça vestibüler organları forseps kopuk emin olmak için tampon etrafında forseps girdap. Bu stereomicroscope altında forseps getirerek durumda kontrol edin. <ol><li> Mikrotübül kapağındaki vida ve hemen sıvı nitrojen içinde örnek dondurma. </li></ol></li></ol> 4. RNA Ekstraksiyonu ve RT-PCR <ol><li> Üreticinin talimatlarına veya tercih edilen laboratuvar protokollerine göre mesajcı RNA (mRNA) ekstre standart yöntemleri takip edin. NOT: mRNA küçük verim yalıtmak için taşıyıcı RNA&#39;lar kullanılan bir ticari kit bu kullanıldıprotokol. Alt elüsyon hacimleri mRNA nihai konsantrasyonlarını arttırmak için kullanılabilir. NOT: Negatif &quot;hayır enzim&quot; denetimleri (UEM) ve &quot;hayır DNA şablonu&quot; denetimleri (NTSC) da gözlenen etkilerin geçerliliğini sağlamak için tamamlanması gereken. </li><li> Hedef genlerin 21-23 tamamlayıcı DNA (cDNA) ve büyütme için mRNA&#39;nın ters transkripsiyon standart yöntemler uygulanır. </li></ol>

<ol>
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Yazarlar hiçbir rakip mali çıkarlarını var beyan.

Burada açıklanan temsili sonuçlar NIR ışık kısa transkraniyal dağıtım (5 gün boyunca 90 sn / gün) plasebo ile tedavi edilen fareler ile karşılaştırıldığında, daha yaşlı farelerin antioksidan ifade seviyelerini yükseltmek için yeterli olduğunu göstermektedir. Nir yaydığı ısı bizim ölçüm cihazı &lt;0,2 ° C 90 sn bitmişti LED ve gibi neden düşüktür - yayılan ısı mitokondriyal ve / veya nöronal aktivasyon kaynağı temsil edebilir ederken, sıçan vestibüler için bildirilen <su...

Denge performansını Azalan ve sonraki düşüşler yaygındır ve doğal yaşlanma 1 maalesef sıklıkla tanımlayan özellikler. Bu düşüşün etkisi fiziksel hem de sosyal olmak, ve önemli ölçüde yaşlı insanlar için yaşam kalitesini düşürür yapabilirsiniz. Buna karşılık, fizik tedaviler ve rehabilitasyon düşme içine araştırma odağı olmuştur ama tekrarlanan düşme yaygınlığı tutarlı azalma ile ilişkili değil. Aynı zamanda, iş periferik veya santral vestibüler sistemi (dengesini korumak için sorumlu sistem) kıt olduğu ve bu sistemleri hedefleyen potansiyel tedavi stratejileri değişiklik ve sınırlı dengesizliğin altında yatan nedenleri araştırmak. Yaşla ilgili maküler dejenerasyon, 2-4, Alzheimer hastalığı modelleri 5-8, ve Parkinson hastalığı dahil olmak üzere, 9-12 yaş ile ilişkili nörodejeneratif hastalıkların üzerine yapılan son çalışmalar si nöro-koruyucu etkiler göstermiştiryakın kızılötesi (NIR) ışık mple non-invaziv bir uygulama. Bundan başka, denge sisteminin, NIR in vitro 13 vestibüler birincil aferent nöronların etkinliğini geliştirmek için kullanılmıştır. Nir ışık mekanizması iyi anlaşılamamakla birlikte, Nir kullanarak çalışmaların çoğu Nir hücresel metabolizmayı kolaylaştırmak için mitokondri karmaşık IV (sitokrom-c oksidaz) 14-17 uyardığını ileri sürmüşlerdir. Vestibüler duyu epitel tip I saç hücrelerinin subkutiküler plaka mitokondri 18 yoğun olduğu ve gibi terapötik Nir tedavisi için bir eylem siteyi temsil edebilir. Burada, transcranially kısa bir invazif olmayan bir tedavi rejimi hücresel metabolizmayı ölçmek için kullanılabilir (ve dolaylı mitokondriyal fonksiyonu olarak), fare vestibüler duyusal epitel anlatılan NIR uygulanır. Ayrıca ele alınan vestibüler duyusal epitel bir preparattır ve NIR bir ubiquito ekspresyonunu arttırdığı gösterilmiştirBize Anti-oksidanın, duyusal epitel (süperoksit dismutaz 1) - daha önce koklea saç hücre hayatta kalma 19 için önemli olduğu gösterilmiştir.

<table><tbody><tr><td>Quantum WARP 10</td><td>Quantum Devices</td><td>2070N030-A</td><td></td></tr><tr><td>Screw top microtubules</td><td>Quality Scientific Plastics</td><td>520-GRD-Q</td><td></td></tr><tr><td>Ketamine</td><td>Parnell, Alexandria Australia</td><td></td><td></td></tr><tr><td>Standard pattern scissors</td><td>FST</td><td>14001-12</td><td></td></tr><tr><td>Carbon steel surgical blades #22</td><td>Livingstone</td><td>SBLDCL 22</td><td></td></tr><tr><td>Friedman-Pearson rongeurs</td><td>FST</td><td>16221-14</td><td></td></tr><tr><td>Stereo microscope</td><td>Leica Microsystems</td><td>A60S</td><td></td></tr><tr><td>Dumont #5 SF forceps</td><td>FST</td><td>11252-00</td><td></td></tr><tr><td>Isolate II RNA Micro Kit</td><td>Bioline</td><td>BIO-52075</td><td></td></tr></tbody></table>

near infrared (nir) light increases expression of a marker of mitochondrial function in the mouse vestibular sensory epithelium

Artan yaş ile denge fonksiyonu düşüş zayıflatmaya yönelik stratejiler denge görevleri ve egzersiz gibi fiziksel tedaviler ağırlıklı odaklanmış bulunmaktadır. Ancak, bu yaklaşım denge düşüş yatan nedenleri ele yoktur. Fareler kullanarak, vestibüler duyu epitel hücrelerin metabolizması üzerine yakın kızılötesi ışığın etkisi (NUR) değerlendirildi. Toplanan veriler bu basit ve güvenli bir müdahale doğal yaşlanma zararlı etkilerinden bu hassas hücreleri koruyan olabileceğini göstermektedir. mRNA izole vestibüler duyusal epiteli (krista ampullaris ve utricular makula) elde edilir ve daha sonra, bir cDNA kitaplığında transkribe edilmiştir. Bu kütüphane, daha sonra her yerde antioksidan (SOD-1) ifadesi için problanmıştır. Antioksidan gen ifadesi hücre metabolizmasını ölçmek için kullanıldı. Genç Nir ve transkranial teslim (4 hafta) ve üzeri kullanma - 5 d (8 9 ay) fareler ve kısa bir tedavi rejimi (90 sn / günays), bu çalışma vestibüler duyusal epitel mitokondriyal fonksiyonu geliştirmek için yeterli olabilir ve yalnız NIR göstermektedir. Vestibüler saç hücre fonksiyonunu artırmak için tedavinin hiçbir mevcut, uygun fiyatlı, non-invaziv yöntemler şu anda olduğundan, dış NIR radyasyon uygulaması vestibüler duyu epiteli inthe hücresel metabolizma üzerinde yaşlanma etkilerini ortadan kaldırmak için potansiyel bir strateji sağlar.

Genç NIR tedavi etkisini (4 hafta) ve daha büyük karşılaştırılması - fareler (8 9 ay) (n = 20) (n = 16), genç antioksidan süperoksit dismutaz 1 (SOD-1) ifadesini ölçülür ve eski plasebo ile tedavi edilmiş, NIR-muamele ya da NIR-bloke fareler. Şekil 2, genç plasebo ile tedavi edilen hayvanlara kıyasla NIR tedavi genç hayvanlarda en fazla 2 misli β-aktin normalize SOD-1 ekspresyonunda önemli bir artış olduğunu göstermektedir (p &lt;0.01) ve genç Nir-bloke hayvanlar (p &lt;0.01)....

Watch this Scientific Journal Video about Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium at JoVE.com

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Mitokondriyal disfonksiyon hücresel yaşlanma bir özelliğidir. Bu kağıt yaşlanma fare vestibüler duyu epitel mitokondriyal fonksiyon geliştirmek için non-invaziv yakın kızılötesi (NIR) tedavi kullanır.

Yakın Kızılötesi (NIR) Işık Fare Vestibüler Duyu Epitelinin Mitokondriyal Fonksiyonun bir Marker Anlatım artırır

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

near-infrared-nir-light-increases-expression-marker-mitochondrial

Research

JoVE Journal

Biology

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

9.9K Görüntüleme.  University of Sydney.  Mitokondriyal disfonksiyon hücresel yaşlanma bir özelliğidir. Bu kağıt yaşlanma fare vestibüler duyu epitel mitokondriyal fonksiyon geliştirmek için non-invaziv yakın kızılötesi (NIR) tedavi kullanır. 

Video: Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

Elektrofizyoloji ve yüksek çözünürlüklü İki foton Mikroskopi için Fare Vestibüler Duyu epiteli bir İzole Yarı sağlam Hazırlık

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

Nörobilim

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

Fırıncı Maya saccharomyces cerevisiae Mitokondriyal Genom İfade Ürünlerinin Etiketlenmesi ve Görselleştirilmesi

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

Biyokimya

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

Üç boyutlu görüntüleme ve mitokondri içinde insan Intraepidermal sinir lifleri analizi

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

Vivo Tayf alanlı optik Koherens tomografi ve lazer Ophthalmoscopy tarama Cx3cr1gfp/gfp muhabir farelerin görüntüleme

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Tıp

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Vitro ve In Vivo insan hücreleri, C. Elegansve fareler Mitophagy tespiti

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

Bir taranabilir<em&gt; İn Vivo</em&gt; Transjenik biyolojik olarak ışık veren kullanarak Mitokondriyal Modülatörleri için tahlil<em&gt; Caenorhabditis elegans</em

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Biyoloji

Measurement of Mitochondrial Membrane Potential In Vivo using a Genetically Encoded Voltage Indicator

Mitochondrial membrane potential (MMP, &#916;&#936;m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen species (ROS) generation, and the import of proteins encoded by the nucleus. Existing methods for measuring &#916;&#936;m typically use lipophilic cation dyes, such as Rhodamine 800 and tetramethylrhodamine methyl ester (TMRM), but these are limited by low specificity and are not well-suited for in vivo applications. To address these limitations, we have developed a novel protocol utilizing genetically encoded voltage indicators (GEVIs). Genetically encoded voltage indicators (GEVIs), which generate fluorescent signals in response to membrane potential changes, have demonstrated significant potential for monitoring plasma membrane and neuronal potentials. However, their application to mitochondrial membranes remains unexplored. Here, we developed protein-based mitochondrial-targeted GEVIs capable of detecting &#916;&#936;m fluctuations in cells and the motor cortex of living animals. The mitochondrial potential indicator (MPI)offers a non-invasive approach to study &#916;&#936;m dynamics in real-time, providing a method to investigate mitochondrial function under both normal and pathological conditions.

This protocol describes the application of mitochondria-targeted genetically encoded voltage indicators (GEVIs). These GEVIs offer a significant advantage over traditional mitochondrial membrane potential dyes by enabling specific, in vivo, and real-time monitoring of mitochondrial membrane potential.

Mitokondriyal Membran Potansiyelinin İn Vivo Olarak Genetik Olarak Kodlanmış Bir Voltaj Göstergesi Kullanılarak Ölçülmesi

Mitochondrial membrane potential (MMP, &#916;&#936;m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen ...

Mitofajinin Ölçülmesi C. elegans ve Karaciğer Kanseri Hücre Hattı

Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, regulated cell death, and the generation of reactive oxygen species (ROS). ROS are vital for key biological processes. However, when uncontrolled, they can lead to oxidative injury, including mitochondrial damage. Damaged mitochondria release more ROS, thereby intensifying cellular injury and the disease state. A homeostatic process named mitochondrial autophagy (mitophagy) selectively removes damaged mitochondria, which are then replaced by new ones. There are multiple mitophagy pathways, with the common endpoint being the breakdown of the damaged mitochondria in lysosomes. 
Several methodologies, including genetic sensors, antibody immunofluorescence, and electron microscopy, use this endpoint to quantify mitophagy. Each method for examining mitophagy has its advantages, such as specific tissue/cell targeting (with genetic sensors) and great detail (with electron microscopy). However, these methods often require expensive resources, trained personnel, and a lengthy preparation time before the actual experiment, such as for creating transgenic animals. Here, we present a cost-effective alternative for measuring mitophagy using commercially available fluorescent dyes targeting mitochondria and lysosomes. This method effectively measures mitophagy in the nematode Caenorhabditis elegans and human liver cells, which indicates its potential efficiency in other model systems.

Yazar Gündemi: Organele Özgü Boyalar Kullanılarak Caenorhabditis elegans ve Memeli Hücrelerinde Mitofajinin Tespiti  

Exploring mitophagy through electron microscopy, genetic sensors, and immunofluorescence requires costly equipment, skilled personnel, and a significant time investment. Here, we demonstrate the efficacy of a commercial fluorescence dye kit in quantifying the mitophagy process in both Caenorhabditis elegans and a liver cancer cell line.

Caenorhabditis elegans ve Memeli Hücrelerinde Mitopajinin Organele Özgü Boyalar Kullanılarak Saptanması

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, ...

Yakın Kızılötesi (NIR) Işık Fare Vestibüler Duyu Epitelinin Mitokondriyal Fonksiyonun bir Marker Anlatım artırır

Özet

Daha Fazla Video Keşfet

Elektrofizyoloji ve yüksek çözünürlüklü İki foton Mikroskopi için Fare Vestibüler Duyu epiteli bir İzole Yarı sağlam Hazırlık

Fırıncı Maya saccharomyces cerevisiae Mitokondriyal Genom İfade Ürünlerinin Etiketlenmesi ve Görselleştirilmesi

Üç boyutlu görüntüleme ve mitokondri içinde insan Intraepidermal sinir lifleri analizi

Vivo Tayf alanlı optik Koherens tomografi ve lazer Ophthalmoscopy tarama Cx3cr1^gfp/gfp muhabir farelerin görüntüleme

Vitro ve In Vivo insan hücreleri, C. Elegansve fareler Mitophagy tespiti

Bir taranabilir

Mitokondriyal Membran Potansiyelinin İn Vivo Olarak Genetik Olarak Kodlanmış Bir Voltaj Göstergesi Kullanılarak Ölçülmesi

Caenorhabditis elegans ve Memeli Hücrelerinde Mitopajinin Organele Özgü Boyalar Kullanılarak Saptanması

Caenorhabditis elegans ve Memeli Hücrelerinde Mitopajinin Organele Özgü Boyalar Kullanılarak Saptanması

Caenorhabditis elegans ve Memeli Hücrelerinde Mitopajinin Organele Özgü Boyalar Kullanılarak Saptanması