We thank the Northwestern University Mouse Histology and Phenotyping Laboratoryfor technical support and assistance, and Noboru Mizushima (University of Tokyo) for providing GFP-LC3 transgenic mice. A. R. and C. H. were supported by the startup funds from Northwestern University and the grant from National Institutes of Health (DK094980).

hayvanları da içeren tüm işlemleri Northwestern University Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylanan kurallara göre gerçekleştirilmiştir. 1. Fare Modelleri <ol><li> egzersiz 8-12 haftalık fareler kullanın. In vivo olarak, egzersiz kaynaklı Otofaji tespit etmek için, görüntüleme çalışmaları ve biyokimyasal analizler için C57BL / 6 fareleri için GFP LC3 transgenik fareler (C57BL / 6 plan) kullanın. </li></ol> 2. Egzersiz kaynaklı Otofaji <ol><li> Koşu bandı kurulumu - zorla egzersiz <ol><li> ACCLIMATE ve 2 gün boyunca 10 ° yokuş yukarı açık koşu bandı üzerinde fareler eğitmek. 1. günde, 5 dakika boyunca fareler egzersiz 8 m / dakika ve 2 gün, 10 m başka bir 5 dakika, ardından 8 m / dakika, 5 dakika boyunca fareler egzersiz / 10 dak. hafif el dürtmek tarafından işletilen fareler teşvik edin. </li><li> 3. günde, fareler 10 ° yokuş yukarı bandı, starti 90 dakika boyunca çalışan tek bir nöbeti maruz izin10 m / dak hızında ng. 40 dakika sonra, 30 dakikalık bir toplam 1 m / dakika, her 10 dk bir oranda bandı hızını artırmak, ve bundan sonra da bir oranda hızını artırmak, 1 m / varıncaya kadar her 5 dakikada en az 17 m / dak, egzersiz süresi 90 dakika ve mesafe çalışan 1.070 metre olmak üzere toplam. </li><li> düşük yoğunluklu aralığında (1.2 Hz) elektrik uyaran ayarlayın ve tekrarlanan ayak çarpmasını önlemek için çalıştırmak için fareler ediyoruz. </li><li> Doku toplanması için, egzersizden hemen sonra servikal dislokasyon tarafından takip izofluran inhalasyon yoluyla fareler, euthanize. Diğer kabul ötenazi yöntemleri aynı zamanda, CO2 ve diğer anestetikler IP enjeksiyon kullanılabilir. otofaji düzeyi ötanazi yöntemleri etkilenecek görünmüyor. </li></ol></li><li> Koşu tekerlek ayarı - istemli egzersiz <ol><li> tek ev sahipliği fare ile bir kafes içinde bir fare çalışan tekerleğini (11,4 cm çap) yerleştirin. </li><li> Bir bisiklet kilometre sayacı kullanarak çalışan kapasitesini doğrulayın.358 de [tekerleğin tam dönme başına (mm) mesafesini] tekerlekli parametresini ayarlayın (11,4 * π). 24 saat sonra koşu mesafeyi ölçün. </li><li> Fare koşu tekerleği ile 2 hafta boyunca gönüllü çalıştıralım. Doku toplanması için, çalışma sonrası sabah fare kurban. </li></ol></li></ol> 3. Otofaji Akı Değerlendirilmesi <ol><li> istirahat ve egzersiz koşulları altında otofajik akının ölçülmesi için, üç gün boyunca Otofaji önleyicisi klorokin ile fareler tedavi etmek ve PBS ile tedavi edilen farelere karşılaştırın. <ol><li> PBS (5 mg / ml) içinde çözülür klorokin ve karın içinden arka arkaya üç gün boyunca 50 mg / kg / gün dozunda farelere chloroquine enjekte edilir. fareler Kurban ve son enjeksiyondan sonra dokular 3 saat toplamak. </li><li> icra fareler için, 50 mg / kg / gün arasında bir dozda birbirini takip eden iki gün klorokin ile ön muamele. Üçüncü gün, sonra, klorokin aynı konsantrasyonda farelere enjekte ve90 dakika onları başka bir 90 dakika koşu bandı üzerinde çalışmasına izin verin. </li><li> servikal dislokasyon tarafından takip izofluran inhalasyon yoluyla fareler, Euthanize. klorokin toplam inkübasyon süresi kontrolü (dinlenme) farelerde (3 saat) aynı şekilde, hemen çalıştırdıktan sonra dokuların toplayın. NOT: Alternatif olarak, fare dokularda otofaji akı belirlemek için klorokin tek bir enjeksiyon kullanın. </li></ol></li></ol> 4. Doku Hasat ve Fiksasyon <ol><li> 7.4 bir son pH PBS: doku hasat öncesi fosfat tamponlu salin (PBS) ve taze% 4 paraformaldehid (gerekli kişisel koruma araçları PFA, DİKKAT) hazırlanması. 4 ° C &#39;de, her iki çözüm saklayın. </li><li> (GFP LC3 fareler için)% 4 PFA, 20 ml - 15 30 ml şırınga doldurun. 20 G kateter iğne ile şırınga bağlayın. </li><li> Egzersiz sonrasında servikal dislokasyon ardından izofluran inhalasyon yoluyla fareler euthanize. </li><li> kendi ba temiz bir çalışma yüzeyine hayvan yerleştirinck. Kesme ve kalp maruz diyaframın keserek göğüs boşluğu açın. </li><li> Akciğer ve karaciğer tamamen soluk açana kadar, kan çıkıp izin karaciğer üzerinde küçük bir kesi yapmak ve kateter yoluyla kalbin sağ ventrikül içine yavaş yavaş 15 ml% 4 PFA toplam enjekte edilir. / Saat 90 ml sabit bir akış oranı ile bir şırınga pompası kullanılarak, idman oturumu hemen sonra fare serpmek. </li><li> Perfüzyon sonra, iğne kaldırmak ve ilgi dokular (örneğin beyin 11 ve iskelet kası) toplamak. iskelet kası için, vastus lateralis teşrih. Kısaca, kuadriseps lokalize, kasları ortaya çıkarmak için geriye doğru bacağın cilt çekin 12 femoris ve femur kemiğinin üst kısmına takılan harici kas Vastus lateralis, teşrih. </li><li> Daha fazla tespit ve dehidrasyon için, 4 ° C&#39;de 24 saat süreyle% 4 PFA GFP LC3 farelerin dokuları yerleştirin ve sonra% 15 aktarmak4 ° C &#39;de sükroz PBS gece boyunca veya dokuları çökene kadar, ve daha sonra gece boyunca 4 ° C&#39;de% 30 sukroz-PBS ya da daha uzun süre depolama için. onlar güçlü ışığa duyarlı olabileceği gibi mümkün olduğunca karanlık örnekleri tutun. </li><li> Western blot analizi için sıvı azot içinde doğrudan C57BL / 6 farelerinden alınan doku dondurma, ve -80 ° C&#39;de saklayın oturur. </li></ol> GFP-LC3 puncta 5. Görüntü Analizi <ol><li> doku süreci <ol><li> Bu etiketlenmiş bir küçük bir petri birkaç dakika &quot;optimal kesme ısısı bileşiği&quot; (OCT) halinde gömme orta dokular (daha önce PFA sabit ve% 30 sukroz içinde saklanan) ön-muamele. </li><li> doku karşılamak için yeterli taze OCT içeren etiketli cryomold doku aktarın. Orient cryomold altına doğru kesit yüzeyi (vastus için kesitte hemi-beyin için kas ve sagital bölümü lateralis). kabarcıkların oluşumunu önlemek. </li><li> freEkim beyaza dönene kadar, kuru buz dolu bir kapalı köpük soğutucu bir kaç dakika cryomold koyarak örnekleri Eze. Etiketli folyo bireysel örnekleri sarın bir plastik torba içinde üzerleri mühürlenir, -80 ° C gecede veya daha uzun saklayın. </li><li> slaytlar üzerinde bölümleri, 10 um kadar bir kalınlıkta Örnek bölümleri kesilmiş ve monte etmek için bir kriyostat kullanarak. -20 ° C&#39;de slaytlar Mağaza ve her zaman mümkün olduğunca ışıktan uzak slaytlar tutun. </li></ol></li><li> slayt hazırlama <ol><li> dondurucu slaytları çıkarın ve oda sıcaklığında onları Çözülme. DAPI ile medya montaj bir damla doku bölümü kapsamaktadır. en uygun büyüklükte bir lamel yerleştirin ve kabarcık oluşumunu önlemek. </li><li> , Lameller kenarları mühür oje, oda sıcaklığında karanlıkta kurumasını bekleyin ve sonra 4 ° C&#39;de bir ışık geçirmez bir kapta slaytlar saklamak veya GFP-LC3 puncta görüntüleme yakalama devam etmek oje kullanın. </li></ol></li><li> &lt;GFP strong&gt; miktar tayini - floresan mikroskop ile LC3 puncta <ol><li> Bütün numuneler için aynı durumda resim (pozlama ve ayarları) yakalayan Epifloresans mikroskopi gerçekleştirin. GFP için, 525 nm&#39;lik bir emisyon dalga boyu kullanımı; DAPI için, 490 nm kullanımı. vastus lateralis bir 60x objektif ve beynin ön korteks bölgesini kullanarak örnek başına en az on görüntüler elde. </li><li> Deney grupları için kör istatistiksel analiz için her görüntüde 2.500 mikron 2 bir doku alanı, GFP-LC3 puncta sayısını ölçmek. &quot;Nesne Sayma&quot; otomatik ölçüm fonksiyonunu kullanarak, her numunenin 10 görüntüleri ortalama sayısını hesaplayın. vastus için Ayarlar kas vardır lateralis: Eşik L = 10, H = 200; 2X pürüzsüz; 1X, temiz ve beyin frontal korteks için ayarları şunlardır: Eşik L = 11, H = 200; 2X pürüzsüz; 1X temiz. </li></ol></li></ol> Otofaji Belirteçleri 6. Western Blot Analizi <ol><li> &lt;strong&gt; Doku süreci <ol><li> liziz tamponu: 50 mM Tris-HCl; 150 mM NaCI; 1 mM EDTA; % 1 Triton X-100; Proteaz inhibitörü ve fosfataz inhibitörleri (taze ilave edildi). </li><li> C Depolama -80 ° den C57BL / 6 farelerinin doku çıkarın. aşağıdaki işlem öncesinde bir jilet ile küçük parçalar halinde kas dokusu kesilir. (Hemi-beyin için) 800 ul veya numune soğuk lizis tamponu (vastus lateralis için) 500 ul ekleyin. Orta hızda bir homojenizatör ile 4 ° C sıcaklıkta örnek homojenize edilir. </li><li> Tam rotasyon ile 4 ° C &#39;de 1 saat kadar 30 dakika daha homojenleştirilir karışımı lize. </li><li> 4 ° C&#39;de 10 dakika boyunca 12,000 xg&#39;de Homojenat Spin pelet atın ve yeni bir tüp içinde supernatant toplamak. </li></ol></li><li> otofaji analizi <ol><li> üreticinin talimatlarına uygun olarak BCA protein tahlil kiti kullanılarak doku lysate&#39;lerin protein konsantrasyonu belirlenir. Her bir örnek normalizeAynı konsantrasyona seyreltildi. </li><li> Bir 1 2x Laemmli numune tampon maddesi ile örnek birleştirin: 1 oranında, 5 95 ° C &#39;de kaynatın - 10 dakika, ve P62 (anti-P62 antikoru gibi, Otofaji Batı benek analizi 14, 13 işaretleri ile devam edin 1: 500 seyreltme) ve LC3 (anti-LC3 antikoru, 1: 500 seyreltme). Buz üzerinde daha uzun kuluçka LC3 birden fazla bozulmuş bantları oluşturur edilebilir hemen örnek tamponu ilave edildikten sonra numune kaynatılır. </li><li> ImageJ kullanarak dansitometre analizi yolu ile P62 ve LC3 bantları sayısal olarak ve ilgili aktin bandının değer normalize. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Otofaji enerji sağlar ve sitoplazma bileşenleri veya zarar görmüş organellerin lizozomal degradasyonu ile sitotoksisitesini azaltır katabolik işlemdir. Otofaji incelenmesi hücresel homeostazının düzenlenmesi ve stres yanıtı mekanizmalarını anlamak için önemlidir. Yeni modeller ve yöntemler çok sayıda patolojik süreçler 16, 17 nasıl katkı bozulmuş otofaji incelemek için, araştırma alanında 15 ortaya çıkıyor....

Otofaji örneğin açlık ve hipoksi, 1, 2 gibi çeşitli stres koşullarına karşılık olarak indüklenir evrimsel olarak korunmuş bir parçalanma yolu vardır. Otofaji sırasında, çift zar kabarcıklar, gereksiz veya hasarlı hücre içi bileşenleri birleştirmek ve bozulması 3 lizozomlar içine taşımak, autophagosomes çağırdı. Bazal Otofaji nörodejenerasyon, tümör oluşumu ve tip 2 diyabet 4, 5, 6 dahil olmak üzere birçok hastalıkta implike olduğu hücresel fonksiyon ve organizmanın gelişimi, ve bozulmuş bazal Otofaji için gereklidir. En iyi bilinen fizyolojik otofaji indükleyici açlık olduğunu. Bununla birlikte, iki önemli kısıtlamalara sahiptir. İlk olarak, açlık, örneğin, etkili hayvanlarda Otofaji uyarılması için farelerde besin yasaklaması 48 saat uzun sürdüğüEn organlarda. İkincisi, açlık zorlukla nedeniyle beyinde göreli olarak istikrarlı bir besin kaynağı, beyin Otofaji neden olur. Aslında, birçok ilaç, kan beyin bariyerini geçemez olarak, küçük moleküllü indüklecilerle otofaji indüksiyon tespit etmek de zordur. Bu nedenle, daha iyi bir hastalığın patogenezinde Otofaji aktivasyon fonksiyonunu analiz etmek için, en son egzersiz süresi 7, 8, 9, kısa bir süre içinde Otofaji uyarılması için daha güçlü bir fizyolojik bir yöntem olduğu keşfedilmiştir. açlık ile karşılaştırıldığında, otofaji etkili bir koşu bandı hızlı 30 olarak dakika çalıştırarak indüklenir. Böylece, egzersiz sağlık yararları aracılık ve hastalıkların önlenmesinde otofaji mekanizmasını incelemek için kullanışlı ve güçlü fizyolojik bir yaklaşımdır. LC3 ve P62 dahil olmak üzere, Otofaji aktivitesinin tespit edilmesi için, bir çok protein işaretleri vardır. LC3 (mikrotübül ilişkili protein 1A / 1B-ışık cHain 3) Bir hücre sıvısı protein (IS LC3 I Otofaji indüksiyondan sonra PE (fosfatidiletanolamin) konjüge edilmiş olduğu) oluşturur. PE-lipide LC3 (LC3 II formu) autophagosomal zarları üzerine işe ve GFP ile işaretlenerek autophagosomes görselleştirmek için kullanılabilmektedir. mikroskopta autophagosomes yapılarını noktalı yanına sitosolden translokasyonu Otofaji indüksiyon gösterir. P62 (örneğin, ubikitinasyon proteinleri gibi) Otofaji yüzeyler için bir kargo reseptördür ve de autophagosomes dahil edilir. Protein autophagosomes birlikte lizozomların parçalanır yana, seviyeleri otofaji akının ölçülmesi için kullanılabilir. Burada zorunlu egzersiz (koşu bandı) ve gönüllü egzersiz (tekerlekler çalıştıran) de dahil olmak üzere aerobik egzersiz, tarafından uyarılan farklı fare dokularda Otofaji ölçmek için bu işaretçileri nasıl kullanılacağını gösterir. Aynı prosedürler, diğer indükleyicilerin tedaviden sonra Otofaji in vivo ölçümü uygulanabilir.

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			<td height="21" style="height:21px;width:247px;">Treadmill</td>
			<td style="width:204px;">Columbus Instruments</td>
			<td style="width:195px;">150-RM Exer 3/6</td>
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			<td height="21" style="height:21px;width:247px;">Mouse running wheel</td>
			<td style="width:204px;">Super Pet</td>
			<td style="width:195px;">100079365</td>
			<td>diameter 11.4 cm</td>
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			<td height="21" style="height:21px;width:247px;">Odometer</td>
			<td style="width:204px;">Bell</td>
			<td style="width:195px;">DASHBOARD 100</td>
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			<td style="width:204px;">KD Scientific</td>
			<td style="width:195px;">KDS100</td>
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			<td height="42" style="height:42px;width:247px;">Fluorescence microscope</td>
			<td style="width:204px;">Nikon</td>
			<td style="width:195px;">Model: inverted microscope ECLIPSE</td>
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			<td height="21" style="height:21px;width:247px;">Cryostat</td>
			<td style="width:204px;">Leica</td>
			<td style="width:195px;">CM 1850UV</td>
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			<td height="42" style="height:42px;width:247px;">Homogenizer</td>
			<td style="width:204px;">IKA</td>
			<td style="width:195px;">003737001 / Model: T10 Basic S1</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Chloroquine</td>
			<td style="width:204px;">CAYMAN CHEMICAL COMPANY</td>
			<td style="width:195px;">14194</td>
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			<td height="21" style="height:21px;width:247px;">Parafolmaldehye</td>
			<td style="width:204px;">SIGMA-ALDRICH</td>
			<td style="width:195px;">P6148</td>
			<td>Personal protection equipment required. This product may release formaldehyde gas, a chemical known to cause cancer</td>
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			<td height="21" style="height:21px;width:247px;">Mounting media</td>
			<td style="width:204px;">Vector Laboratories</td>
			<td style="width:195px;">H-1200</td>
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			<td height="21" style="height:21px;width:247px;">p62 antibody</td>
			<td style="width:204px;">BD Biosciences</td>
			<td style="width:195px;">610833</td>
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			<td height="21" style="height:21px;width:247px;">LC3 antibody</td>
			<td style="width:204px;">Novus Biologicals</td>
			<td style="width:195px;">NB100-2220</td>
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		<tr height="21">
			<td height="21" style="height:21px;">2X Laemmli Sample Buffer</td>
			<td style="width:204px;">Bio-Rad Laboratories</td>
			<td style="width:195px;">161-0737</td>
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			<td height="21" style="height:21px;width:247px;">ImageJ</td>
			<td style="width:204px;">NIH</td>
			<td style="width:195px;"></td>
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activating autophagy by aerobic exercise in mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Bu protokol aerobik egzersiz fare dokularda Otofaji ikna etmek için iki farklı yöntem açıklanır: aklimasyonundan iki gün ilerledi çok şeritli koşu bandı üzerinde zorunlu egzersiz 90 dakika olmak üzere toplam; veya tek ev sahipliği fareler tarafından kullanılan bir koşu tekerlek üzerinde gönüllü egzersiz iki hafta. Her seri protokol, çeşitli organlarda, flüoresans mikroskopisi ve Western blot analizi ile Otofaji akı ölçebilir. <p class="jove_content" fo:keep...

Watch this Scientific Journal Video about Activating Autophagy by Aerobic Exercise in Mice at JoVE.com

Activating Autophagy by Aerobic Exercise in Mice

Otofaji aktivasyonu çeşitli hastalıkların önlenmesinde yararlıdır. Fizyolojik yaklaşımlardan biri in vivo otofaji fiziksel egzersiz teşvik etmek. Burada aerobik egzersiz ile Otofaji etkinleştirmek ve farelerde otofaji düzeylerini ölçmek için nasıl gösterir.

Farelerde Aerobik Egzersiz ile Autophagy etkinleştirme

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

activating-autophagy-by-aerobic-exercise-in-mice

Research

JoVE Journal

Biology

11.7K Görüntüleme.  Northwestern University.  Otofaji aktivasyonu çeşitli hastalıkların önlenmesinde yararlıdır. Fizyolojik yaklaşımlardan biri in vivo otofaji fiziksel egzersiz teşvik etmek. Burada aerobik egzersiz ile Otofaji etkinleştirmek ve farelerde otofaji düzeylerini ölçmek için nasıl gösterir. 

Video: Activating Autophagy by Aerobic Exercise in Mice

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.

This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

Aerobik Mantarlar Kontrollü Bitki Doku Ayrışma Agar-Blok Microcosms

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

Biyoloji

Determining the Contribution of the Energy Systems During Exercise

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given physical activity. Although some sports are relatively easy to be reproduced in a laboratory (e.g., running and cycling), a number of sports are much more difficult to be reproduced and studied in controlled situations. This method presents how to assess the differential contribution of the energy systems in sports that are difficult to mimic in controlled laboratory conditions. The concepts shown here can be adapted to virtually any sport.
The following physiologic variables will be needed: rest oxygen consumption, exercise oxygen consumption, post-exercise oxygen consumption, rest plasma lactate concentration and post-exercise plasma peak lactate. To calculate the contribution of the aerobic metabolism, you will need the oxygen consumption at rest and during the exercise. By using the trapezoidal method, calculate the area under the curve of oxygen consumption during exercise, subtracting the area corresponding to the rest oxygen consumption. To calculate the contribution of the alactic anaerobic metabolism, the post-exercise oxygen consumption curve has to be adjusted to a mono or a bi-exponential model (chosen by the one that best fits). Then, use the terms of the fitted equation to calculate anaerobic alactic metabolism, as follows: ATP-CP metabolism = A1 (mL . s-1) x t1 (s). Finally, to calculate the contribution of the lactic anaerobic system, multiply peak plasma lactate by 3 and by the athlete&#x2019;s body mass (the result in mL is then converted to L and into kJ).
The method can be used for both continuous and intermittent exercise. This is a very interesting approach as it can be adapted to exercises and sports that are difficult to be mimicked in controlled environments. Also, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study.

This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.

Egzersiz sırasında Enerji Sistemlerinin Katkısı Belirlenmesi

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

Farelerde Hidrodinamik Gen Taşıyıcı Proteinlerin Geçici İfadesi

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

Otofaji Analiz<em&gt; Penicillium chrysogenum</em&gt; Floresan Mikroskopi ile Kombinasyon Açlık Pedleri kullanarak

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fekal Glukokortikoid Analizi: Tek tırnaklılarda in Non-invaziv Adrenal İzleme

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

Protein-protein etkileşimlerinin Autophagy araştırma çalışma

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

Otofajinin Kas Hücrelerinde Yerinde İmmunofloresan Boyanması

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

Laktat dehidrogenaz haciz tahlil — Toplu Autophagic haciz etkinliğini memeli hücreleri belirlemek için basit ve güvenilir Yöntem

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman&#39;s space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)-dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman&#39;s space and all segments of the nephron within the imaging depth limit, about 100 &#181;m below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman&#39;s space. We expect this technique to have wide applicability in renal physiologic investigation.

We present use of 2-photon microscopy to place a micropipette within Bowman&#39;s urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

Micropuncture tarafından 2 foton mikroskobu kolaylaştırdı farelerde Bowman'ın alanı

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional ...