The Fralin Life Science Research Institute and The Metabolic Phenotyping Core at Virginia Tech supported this work.

Hayvan çalışmaları Kurumsal Hayvan Bakım tarafından onaylanmış bir protokol çerçevesinde gerçekleştirilen ve Virginia Politeknik Enstitüsü ve Eyalet Üniversitesi&#39;nde Committee bulundu. 1. Kurulum (Saat: ~ 45 dk) <ol><li> 37 ° C su banyosunda Mitokondri (IBM) 1 ve IBM2 için% 0.25 tripsin, İzolasyon Tampon çözülme dondurulmuş saklar. </li><li> Cam ve yüksek saflıkta su ile,% 70 etanol içinde kesme durulayınız. </li><li> 4 bölümden IBM1 1 kısım tripsin seyrelterek% 0.25 tripsin stokundan% 0.05 tripsin çözüm hazırlayın. </li><li> Vidalı kapaklı 1.5 ml mikrosantrifüj tüpü içinde (100 oranı 1) hücre liziz tamponu ile proteaz ve fosfataz inhibitörü kokteyl karıştırın. </li><li> Kısım 5 ml 15 ml&#39;lik plastik tüp içine% 0.05 tripsin (5 ml / fare). </li><li> Set Motor 80 RPM doku homojenleştirici tahrik. </li></ol> İskelet Kası Mitokondri 2. izolasyonu (Saat: ~ 90 dk) <ol><li> CO 2 ile bir fare Euthanize </suservikal dislokasyon b&gt; inhalasyon. </li><li> Aşağıdaki adımlarda açıklandığı gibi soleus (~ 75-100 mg toplam) içeren kuadriseps ve gastroknemius kasının, kırmızı kas kaldırın: <ol><li> Fare doğru cilt soyma. </li><li> Kuadriseps kökenli noktası üzerinde yağ yastığı çıkarın. Ince uçlu makasla patella bağlı olduğu kuadriseps tendonu kesin. Not: quadriceps proksimal femur ön kısmı üzerindeki anatomik pozisyonuna ile tanımlanır. </li><li> Kemik kuadriseps kurtarmak için, femoral arter kaçınarak Yavaşça, kemik ve kuadriseps arasındaki künt snip. Kuadriseps kas özgürleştirmek ve soğutulmuş PBS kuadriseps kas yerleştirmek için femur üzerinde orijin noktasında ince uçlu makasla tendon kesin. </li><li> Makasla kuadriseps üzerinde görünür yağ dokusu çıkarın. Yani femur örten oldu kas kısmı u bakacak şekilde çevirin üzerinde kuadrisepss. Bir yelpaze hareket forseps ile kuadriseps kas açın. Soğutulmuş IBM1 5 ml içeren bir cam kapta ince uçlu makas ve yer ile her lobdan iki görünür kırmızı kas kısımlarını çıkarın. Not: kuadriseps her lobun yan kenarında bulunan kırmızı bir kas şeridi ile iki lob olarak görünür. </li><li> İnce uçlu makasla Aşil tendonu örten deri kesin ve fare doğru cilt soyma. Maruz Aşil tendonu kesin ve fare gövdesine doğru kas soyun. </li><li> Gastroknemius serbest ve soğutulmuş PBS kendi tendon bağlı gastrocnemius yerleştirmek için ince uçlu makasla femur lateral ve medial kondil de tendon kesin. </li><li> Üzerinde gastroknemius ve soleus kas çevirin kalkmasına ve soğutulmuş IBM1 5 ml içeren beher içine yerleştirin. Fan gastroknemius açın. Ince uçlu makas ile üç görsel kırmızı kas kısımlarını (iki yanal ve bir medial yüzeysel kırmızı şeritler) çıkarıns ve soğutulmuş IBM1 5 ml ihtiva eden bir behere aktarın. </li></ol></li><li> (Adım 2.2 de tarif edildiği gibi) fare diğer bacak kırmızı kas diğer ~ 75-100 mg çıkarın ve proteaz ve fosfataz inhibitörü kokteyli ve hücre liziz tamponu (50 mM Tris-HCl, 1 ile 1.5 ml mikrosantrifüj tüpü içine yerleştirmek mM EDTA, 150 mM NaCl,% 1 sodyum dodesil sülfat,% 0.5 sodyum deoksikolat,% 1 polioksietilen (9) nonilfenileter. hemen Western blotlama daha sonra kullanmak için sıvı azot içinde bu ikinci örnek flaş dondurularak dallanmış (adım 6.1). </li><li> Büyük bir kova buz üzerinde önceden soğutulmuş, düz plastik yüzeyi yerleştirin. Plastik yüzeye IBM1 bir damla dökün ve IBM1 damlacık içinde bölümlere kırmızı kas (adım 2.2.4 ve 2.2.7) tüm yerleştirmek için cımbız kullanın. Değişen jilet ile 3 tek kenar jilet her 40 saniye ile 2 dakika süreyle kas dokusu kıyma. </li><li> Th plastik yüzey tutarak taze IBM1, 5 ml ile yeni bir behere kıyılmış doku transfere beher ve bir jilet ile beher içine kas kazıma. Bu çözümü alın ve 50 ml konik tüp yerleştirilir 100 mikron hücre süzgecinden boşaltın. </li><li> Bir hassas bir görev silecek ile doku Blot ve daha sonra% 0.05 tripsin solüsyonu 5 ml aktarın. Görev silecek doku kaldırmak için cımbız kullanın. </li><li> % 0.05 tripsin solüsyonu içinde 30 dakika için buz üzerinde kas dokusu inkübe edin. </li><li> 4 ° C&#39;de 3 dakika boyunca 200 x g&#39;de 15 ml konik tüp içeren tripsin / kas karışımı dönerler. </li><li> Bir atık kabına tripsin süpernatant dökün ve buz 3 ml soğuk IBM1 ile pelletini. 45 ml&#39;lik bir cam homojenizatör tüp doku aktarın. Kalan numune toplamak ve cam homojenleştirici tüp bu eklemek için başka bir 1.5 ml IBM1 ile 15 ml konik tüp durulayın. </li><li> Cam homogenizer beher içinde minimal hareket yani buz ile bir bardak ya da plastik kap dolu yarım içine cam homojenizatör tüp yerleştirin. </li><li&gt; 80 rpm&#39;de 10 geçişle bir motor tahrikli doku homojenleştirici bağlı PTFE havan ile doku / IMB1 homojen bir karışım. ~ 2 saniye, her geçişte alt tutun. </li><li> Yeni 15 ml konik tüp doku Homojenat aktarın ve IBM1 6.5 ml cam homojenizatör tüp durulayın. Doku Homojenat içeren 15 ml konik tüp içine IBM1 dökün. </li><li> 4 ° C&#39;de 10 dakika boyunca 700 g&#39;de 15 ml konik bir tüp dönerler. Yavaşça cam yüksek mukavemetli santrifüj tüpüne süpernatant dökün ve pelet atın. </li><li> 4 ° C&#39;de 10 dakika boyunca 8000 x g&#39;de, yukarıdaki aşamada elde edilen süpernatant dönerler. </li><li> IBM1 Süpernatantı ve yavaşça IBM2 500 ul ekleyerek pelet yeniden askıya. Bir pipet ucunu kesti ve hafifçe karıştırma ve hareketleri karıştırılarak IBM2 pelet homojenize. Pelet tamamen askıya sonra karışıma IBM2 başka 4,5 ml ilave edilir. T gibi büyük pelet parçaları kırarak aşırı öğütme kaçının: Notonun mitokondriyal zarlarının zarar verebilir. </li><li> 4 ° C&#39;de 10 dakika boyunca 8000 x g&#39;de IBM2 / doku Homojenat dönerler. Not: Bu adım, toplam protein içeriğinin katkıda bulunabilir bakiye BSA yana Aşama 4 izole mitokondri daha doğru bir protein ölçümü için temiz bir yüksek mukavemetli santrifüj tüpüne tekrarlanabilir. </li><li> Nazikçe Süpernatantı ve kaldırmak, ancak tamamen kesilmiş olan nokta ile bir pipet ile IBM2 iki 25 ul artışlarını ekleyerek pelet yeniden askıya. Yavaşça karıştırın ve IBM2 25 ul her bir ilaveden sonra pelet karıştırın. Buz üzerinde bu mitokondriyal stok yerleştirin. </li></ol> Tüm Doku Lizat 3. Homojenizasyon (Saat: ~ 45 dakika; Adım sırasında Yapılabilir 7) <ol><li> Bu adım 2.3 sıvı azot içine yerleştirilmiştir gelen kas çıkarın ve tam çözülmüş kadar oda sıcaklığında inkübe edin. </li><li> Ince uçlu makasla buz üzerinde ~ 30 saniye için doku kıyma. </li><li> Zirco iki 100 ul kepçe eklemekıyılmış doku içeren adım 3.2 vidalı kapaklı 1.5 ml mikrosantrifüj tüp uranyum Oksit Boncuk. 4-6 (orta ayar) bir hız ayarında iki 5 ila üç dakika döngüleri kullanarak bir boncuk değirmeni doku homojenleştiricide doku homojenize. </li><li> 4 ° C&#39;de 10 dakika boyunca 12.000 x g&#39;de aşama 3,3 karışımın dönerler. 1.5 ml mikrosantrifüj tüp içine spin ve transferden sonra 1000 ul pipet kullanarak süpernatantı. Pelet atın ve buz üzerinde supernatant yerleştirin. </li></ol> 4. Protein Tayini (süre: ~ 30 dakika) <ol><li> Üreticinin talimatlarına göre BCA Protein tahlil kiti kullanılarak tüm doku lizat (adım 3.4) ve mitokondriyal stokunun (adım 2.17) protein konsantrasyonu belirleyin. </li></ol> 5. Mitokondri Membran Bütünlüğü; Sitrat Sentaz (CS) Etkinlik (Saat: ~ 15 dk) <ol><li> Yüksek saflıkta su mitokondriyal stokunun 20 dilüsyonları: iki ayrı 1 yapın. EklemekTek örnek Triton 100 X% 0,1 ve düşük ayarda (765 Watt, 2 genlik) it at ses dalgalarına maruz. Buz üzerinde başka seyreltme bırakın. Sonikasyon sonra buza örneği dönün. </li><li> Olan bir sitrat sintaz deneyi gerçekleştirmek hem sigara soniklendi ve daha önce tarif edildiği gibi 11,12 bir spektrofotometrik deneyle mitokondriyal dilüsyonları sonike edilmiştir. </li><li> Aşağıdaki denklemleri kullanarak sağlam mitokondri zarlarının yüzdesini belirleyin: (Non-soniklendi CS faaliyeti ÷ soniklendi CS aktivitesi) * 100 </li></ol> 100- N (yüzde yukarıdan elde) 6. Mitokondri İzolasyon Kalite; Western Blot (Saat: Lab Protokole göre) <ol><li> Daha önce açıklandığı gibi 12 tam doku lizat ve izole mitokondri Batı blotting gerçekleştirin. Not: GAPDH kullanımlar primer antikor (1: 1000 seyreltme,% 5 BSA / TBS-T) ve COXIV (1: 1000 seyreltme içinde% 5 yağsızsüt / TBS-T, sırasıyla anti-keçi, tavşan sekonder antikor, (1) ve akabinde: 10,000). </li></ol> 7. Respirometrik Deneyi Run (Saat: ~ 90 dk) <ol><li> Daha önce açıklandığı gibi mikroplaka bazlı O 2 tüketimi ölçüm teknolojisi kullanılarak istenilen Respirometrik tahlilleri yapın (arkadaşı makale ve Rogers bkz ark 8). </li><li> Devlet 4o (RCR) tarafından State 3 bölünmesi veya Devlet 4o Devlet 3U bölerek Solunum kontrol oranı (RCR) belirleyin. RCR belirlerken noktadan noktaya izlerinden orta (ortalama) noktası, ya da en yüksek (Devlet 3) ve en düşük fiyatları (Devlet 4o) kullanın. </li></ol>

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George Rogers is an employee of Seahorse Bioscience that produces the instrument for which this protocol was modified.

Burada sunulan yöntemler fare iskelet kası minimal miktarlarda (~ 75-100 mg) bir mitokondriyal izolasyon prosedürü ayrıntılı bir açıklamasını sağlar. Bu izolasyon prosedürü O 2 tüketimi oranları, RCR değerleri, maksimal sitrat sentaz aktivitesi ve immunoblotting protein ifadesi ile kanıtlandığı gibi yüksek işleyişi, saf mitokondri (~ 450 mikrogram) elde edebiliyor. Önemli olarak, bu prosedür izole mitokondri, yüksek verimlilik sağlayan mikroplaka göre O2 tüketimi tekno...

The primary function of mitochondria is to produce ATP from oxidative phosphorylation. However, mitochondria have many other important cellular functions including but not limited to: the production and detoxification of reactive oxygen species, the regulation of cytoplasmic and mitochondrial calcium, organelle trafficking, ionic homeostasis, and involvement in apoptosis1,2. Therefore, it is not surprising that dysfunctional mitochondria play a role in many disease pathologies, such as aging, neurodegenerative diseases, cardiovascular disease, cancer, obesity, and diabetes3,4. Importantly, skeletal muscle mitochondria specifically are involved in many of these pathologies3-5.
Mitochondrial respiration assays using isolated mitochondria allow for the assessment of electron transport chain and oxidative phosphorylation function, and the determination of mechanism(s) of action of drugs and proteins that modulate metabolism. Mitochondrial isolation procedures exist for multiple tissue and cell types for a variety of species6,7. However, these procedures often require large quantities of tissue/cells for a high quality mitochondria yield necessary for classic respirometric assays. 
Microplate based respirometirc assays allow for high throughput measurements using minimal quantities of isolated mitochondria, often just several &#181;g per well8. Therefore, we present a modification of previously published methods7 to allow for high quality mitochondria to be isolated from smaller quantities of mouse skeletal muscle for use in microplate based respirometirc assays. In addition, methods are provided to establish the quality of the mitochondrial isolation preparation and the integrity of the mitochondrial membranes. Given that skeletal muscle mitochondria are involved in many pathological conditions, the measurement of O2 consumption in mechanistically driven studies is becoming more prevalent in biomedical research9,10.

<table><tbody><tr><td>Essentially Fatty</td><td>Sigma Aldrich</td><td>A6003</td><td>N/A</td></tr><tr><td>Acid Free- BSA</td><td></td><td></td><td></td></tr><tr><td>Tris/HCl</td><td>Promega</td><td>H5123</td><td>N/A</td></tr><tr><td>KCL</td><td>Sigma Aldrich</td><td>P9541</td><td>N/A</td></tr><tr><td>Tris Base</td><td>Promega</td><td>H5135</td><td>N/A</td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>E6511</td><td>N/A</td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E4378</td><td>N/A</td></tr><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>S7903</td><td>N/A</td></tr><tr><td>D-Mannitol</td><td>Sigma Aldrich</td><td>63559</td><td>N/A</td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Thermo Scientific</td><td>25200-056</td><td>N/A</td></tr><tr><td>Sodium Chloride&lt;br/&gt; White Crystals or Crystalline Powder&lt;br/&gt; &amp;ge;99.0 %</td><td>Fisher Scientific</td><td>BP3581</td><td>N/A</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma Aldrich</td><td>L3771&amp;nbsp;</td><td>N/A</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma Aldrich</td><td>D6750&amp;nbsp;</td><td>N/A</td></tr><tr><td>Polyoxyethylene (12) nonylphenyl ether, branched</td><td>Sigma Aldrich</td><td>238651</td><td>N/A</td></tr><tr><td>Single Edge Razor Blades</td><td>Fisher Scientific</td><td>12-640</td><td>N/A</td></tr><tr><td>Falcon- 100 uM Nylon Cell Strainers</td><td>Fisher Scientific</td><td>352360</td><td>N/A</td></tr><tr><td>Halt Protease &amp;amp; Phosphatse Inhibitor Cocktail</td><td>Thermo Scientific</td><td>1861284</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes with screw cap</td><td>Thermo Scientific</td><td>3474</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Zirconium Oxide beads</td><td>Fisher Scientific</td><td>C9012112</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>GAPDH antibody (1D4)</td><td>Santa Cruz Biotechnology</td><td>sc-59540</td><td>N/A</td></tr><tr><td>Anti- COXIV antibody</td><td>Cell Signaling</td><td>4844s</td><td>Any mitochondrial inner membrane protein will suffice</td></tr><tr><td>Peroxidase conjugated affinipure Donkey, Anti Rabbit IgG (H+L)</td><td>Jackson ImmunoResearh</td><td>711-035-152</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Peroxidase conjugated affinipure Goat, Anti Mouse IgG (H+L)</td><td>Jackson ImmunoResearh</td><td>115-001-003</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Triton-X100</td><td>Sigma Aldrich</td><td>X100</td><td>N/A</td></tr><tr><td>Pierce BCA Protein Assay Kit&amp;nbsp;</td><td>Thermo Scientific</td><td>23225</td><td>N/A</td></tr><tr><td>Pyruvic Acid, 98%</td><td>Sigma Aldrich</td><td>107360</td><td>Store at 4 &amp;deg;C,pH to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td>Succinic Acid</td><td>Sigma Aldrich</td><td>S9512</td><td>Store at room temperature, pH to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td>L(-) Malic Acid, BioXtra, &amp;ge;95%</td><td>Sigma Aldrich</td><td>M6413</td><td>Store at room temperature, to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td>L-Glutamic acid</td><td>Sigma Aldrich</td><td>G1251</td><td>Store at room temperature, to 7.4 with KOH prior to use in respirometric assay, to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Palmitoyl L-carnitine chloride</td><td>Sigma Aldrich</td><td>P1645</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Oligomycin A, &amp;ge; 95% (HPLC)</td><td>Sigma Aldrich</td><td>75351</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Carbonyl cyanide 4-(trifluoromethoxy)</td><td>Sigma Aldrich</td><td>C2920</td><td>Store at 2-8 &amp;deg;C</td></tr><tr><td>phenylhydrazone</td><td></td><td></td><td></td></tr><tr><td>&amp;ge;98% (TLC), powder [FCCP]</td><td></td><td></td><td></td></tr><tr><td>Antimycin A from streptomyces sp.</td><td>Sigma Aldrich</td><td>A8674</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Adenosine 5&amp;prime;-diphosphate monopotassium salt dehydrate [ADP]</td><td>Sigma Aldrich</td><td>A5285</td><td>Store at -20 &amp;deg;C, to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Rotenone</td><td>Sigma Aldrich</td><td>R8875</td><td>Store at room temperature</td></tr></tbody></table>

isolation of mitochondria from minimal quantities of mouse skeletal muscle for high throughput microplate respiratory measurements

Fonksiyonel iskelet kası mitokondri yaşlanma, obezite ve Tip II diyabeti olan gözlenen değiştirilmiş metabolizmasında rol oynar. İzole mitokondriyal müstahzarlardan mitokondriyal respirometrik deneyler, ilaç ve metabolizmasını modüle eden proteinlerin hareket mekanizması (ler) in mitokondriyal fonksiyonun değerlendirilmesi, hem de belirlenmesi için izin verir. Güncel izolasyon prosedürleri genellikle Respirometrik deneyleri için gerekli yüksek kaliteli mitokondri verim dokusunun büyük miktarlarda gerektirir. Bu tarifnamede sunulan yöntemler (~ 450 ug), yüksek verimli, solunum ölçümlerde kullanmak için, fare, iskelet kası, minimum miktarlarda (~ 75-100 mg) izole edilebilir ne kadar kaliteli saflaştırılmıştır mitokondri tarif eder. Bizim izolasyon yöntemi spektrofotometrik sitrat sentaz aktivitesini ölçerek 92.5 ± 2.0% bozulmamış mitokondri verimleri belirlenmiştir. Buna ek olarak, izole edilmiş bir mitokondride Western blot analizi, cytoso soluk ekspresyonu ile sonuçlandılic proteini, GAPDH ve mitokondriyal protein, COXIV sağlam ifadesi. İzole mitokondri belirgin bir GAPDH bantın yokluğu, izolasyon prosedürü sırasında olmayan mitokondriyal kaynaklardan çok az kirlenme göstergesidir. En önemlisi, O 2 tüketimi mikro plakalı tabanlı teknoloji ile hızı ve yüksek birleştiğinde gösterir akuple Respirometrik deneyleri için solunum kontrolü oranı (RCR) belirlenmesi ölçümü (RCR, tüm tahliller için&gt; 6) ve fonksiyonel mitokondri. Sonuç olarak, ayrı bir kıyma adım eklenmesi ve önemli ölçüde daha önce bildirilen yöntemin motor tahrikli homojenleştirme hızını azaltarak yüksek fonksiyonlu teneffüs yüksek bağlanmış mitokondri sonuçlanır fare iskelet kası küçük miktarlarda yüksek kalitede ve saflaştırılmış mitokondri izolasyonunu sağladı mikro-bazlı respirometirc deneyleri sırasında.

Sitrat sintaz aktivitesi sitrat sentez intakt membranlı mitokondri süspansiyonlar içinde mevcut olmamalıdır böylece iç mitokondriyal zarın içinde bulunan ve bu yana membran bütünlüğü için bir ölçüt olarak hizmet vermektedir. 1 sonike ile karşılaştırıldığında sigara sonike mitokondriyal örnekleri sitrat sentaz aktivitesini temsil etmektedir Şekil Aynı izolasyondan örnekler. Sitrat sintaz aktivitesi istatistiksel olarak önemli bir artış mitokondri sonuçla...

Watch this Scientific Journal Video about Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements at JoVE.com

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Yüksek Verimli Mikroplate Solunum Ölçümler için Fare İskelet Kası Minimal Miktarları gelen Mitokondri İzolasyonu

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

isolation-mitochondria-from-minimal-quantities-mouse-skeletal-muscle

Research

JoVE Journal

Biology

12.7K Görüntüleme.  Virginia Tech. Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Video: Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Yüksek verim Mikroplate Solunum Ölçümler Fare İskelet Kası Minimal Miktarları gelen kondriyayı İzole kullanma

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Gelişim Biyolojisi

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.

Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function. Therefore, protein import serves as a barometer of cellular health.

İskelet Kas Mitokondrilerinin Protein İthalat Kapasitesinin Ölçümü

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, ...

Biyokimya

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Perkütan İğne Biyopsisi ile Elde Edilen İskelet Kası Doku İzole Mitokondri hazırlanması ve Respirometrik Değerlendirmesi

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Tıp

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Yetişkin ve Embriyonik İskelet, Kas Microexplant Kültür ve İskelet Kası Kök Hücre İzolasyonu

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Biyoloji

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

İskelet Kası Mitokondrial İzolasyon

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

İskelet Kası progenitörlerin saflaştırılması

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

<meta charset="utf8"></head><body>Yüksek Kaliteli Biyoenerjetik Çalışmalar için Kemirgenlerden Mitokondriyal İzolasyon

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

<meta charset="utf8"></head><body>Yazar Gündemi: Mitokondriyal Kalsiyum Fosfatın Kalp Yetmezliği ve Biyoenerjetik Üzerindeki Rolünü Ortaya Çıkarmak

Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution respirometry.

Kemirgen Kalp Dokusundan Sağlam Mitokondriyal İzolasyon 

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

Mitokondri İzole Fare Heart Geçirgenliği Geçiş Gözenek Açma Multi-parametre ölçümü

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Yüksek Verimli Mikroplate Solunum Ölçümler için Fare İskelet Kası Minimal Miktarları gelen Mitokondri İzolasyonu

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Yüksek verim Mikroplate Solunum Ölçümler Fare İskelet Kası Minimal Miktarları gelen kondriyayı İzole kullanma

İskelet Kas Mitokondrilerinin Protein İthalat Kapasitesinin Ölçümü

Perkütan İğne Biyopsisi ile Elde Edilen İskelet Kası Doku İzole Mitokondri hazırlanması ve Respirometrik Değerlendirmesi

Yetişkin ve Embriyonik İskelet, Kas Microexplant Kültür ve İskelet Kası Kök Hücre İzolasyonu

İskelet Kası Mitokondrial İzolasyon

İskelet Kası progenitörlerin saflaştırılması

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Kemirgen Kalp Dokusundan Sağlam Mitokondriyal İzolasyon

Kemirgen Kalp Dokusundan Sağlam Mitokondriyal İzolasyon

Mitokondri İzole Fare Heart Geçirgenliği Geçiş Gözenek Açma Multi-parametre ölçümü