Name: evaluation of injury-induced senescence and in vivo reprogramming in the skeletal muscle
Uploaded: 2017-10-26T14:00:00
Description: Watch this Scientific Journal Video about Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle at JoVE.com

Biz onun mükemmel teknik destek için Clemire Cimper için borçlu bulunmaktadır. H.L. laboratuvar çalışmalarında Institut Pasteur, Merkezi Ulusal pour la Recherche bilimsel ve Agence Nationale de la Recherche (Laboratoire d'Excellence Revive, Investissement d'Avenir; tarafından finanse edildi ANR-10-LABX-73), Agence Nationale de la Recherche (ANR-16-CE13-0017-01) ve Fondation ARC (PJA 20161205028). C.C. ve A.C. doktora ve doktora sonrası Burslar canlandırmak Konsorsiyumu üzerinden tarafından finanse edilmektedir.

hayvanlar ele Avrupa toplum kuralları ve Etik Komitesi Institut Pasteur (CETEA) onaylı kurallarının başı olarak.
 1. hisse senedi çözümleri hazırlıkları <ol><li>kas örnek fiksasyon için malzemeleri hazırlayın. 0.5 g tragacanth sakız kas fiksasyon için donma katıştırma orta yapmak RT 20 mL su ile çözülür.</li>
	<li>SA-β-Gal boyama için çözümleri hazırlayın.
	<ol><li>Hisse senedi çözümleri K 3 Fe(CN) 6 (100 mM), K ...

<ol>
	<li>Munoz-Espin, D., Serrano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+senescence:+from+physiology+to+pathology.">Cellular senescence: from physiology to pathology.</a> Nat Rev Mol Cell Biol. 15 (7), 482-496 (2014).</li><li>Baker, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+occurring+p16(Ink4a)-positive+cells+shorten+healthy+lifespan.">Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan.</a> Nature. 530 (7589), 184-189 (2016).</li><li>Hayflick, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Limited+in+Vitro+Lifetime+of+Human+Diploid+Cell+Strains.">The Limited in Vitro Lifetime of Human Diploid Cell Strains.</a> Exp Cell Res. 37, 614-636 (1965).</li><li>Coppe, J. P., Desprez, P. Y., Krtolica, A., Campisi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+senescence-associated+secretory+phenotype:+the+dark+side+of+tumor+suppression.">The senescence-associated secretory phenotype: the dark side of tumor suppression.</a> Annu Rev Pathol. 5, 99-118 (2010).</li><li>Yun, M. H., Davaapil, H., Brockes, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+turnover+of+senescent+cells+during+regeneration+of+a+complex+structure.">Recurrent turnover of senescent cells during regeneration of a complex structure.</a> Elife. 4, (2015).</li><li>Demaria, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+essential+role+for+senescent+cells+in+optimal+wound+healing+through+secretion+of+PDGF-AA.">An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA.</a> Dev Cell. 31 (6), 722-733 (2014).</li><li>Mosteiro, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+damage+and+senescence+provide+critical+signals+for+cellular+reprogramming+in+vivo.">Tissue damage and senescence provide critical signals for cellular reprogramming in vivo.</a> Science. 354 (6315), (2016).</li><li>Chiche, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injury-Induced+Senescence+Enables+In+Vivo+Reprogramming+in+Skeletal+Muscle.">Injury-Induced Senescence Enables In Vivo Reprogramming in Skeletal Muscle.</a> Cell Stem Cell. , (2016).</li><li>Ritschka, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+senescence-associated+secretory+phenotype+induces+cellular+plasticity+and+tissue+regeneration.">The senescence-associated secretory phenotype induces cellular plasticity and tissue regeneration.</a> Genes Dev. 31 (2), 172-183 (2017).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decade+of+transcription+factor-mediated+reprogramming+to+pluripotency.">A decade of transcription factor-mediated reprogramming to pluripotency.</a> Nat Rev Mol Cell Biol. 17 (3), 183-193 (2016).</li><li>Abad, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+in+vivo+produces+teratomas+and+iPS+cells+with+totipotency+features.">Reprogramming in vivo produces teratomas and iPS cells with totipotency features.</a> Nature. 502 (7471), 340-345 (2013).</li><li>Ohnishi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+Termination+of+Reprogramming+In+Vivo+Leads+to+Cancer+Development+through+Altered+Epigenetic+Regulation.">Premature Termination of Reprogramming In Vivo Leads to Cancer Development through Altered Epigenetic Regulation.</a> Cell. 156 (4), 663-677 (2014).</li><li>Guardiola, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Acute+Skeletal+Muscle+Regeneration+by+Cardiotoxin+Injection.">Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection.</a> J Vis Exp. (119), (2017).</li><li>Le Roux, I., Konge, J., Le Cam, L., Flamant, P., Tajbakhsh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numb+is+required+to+prevent+p53-dependent+senescence+following+skeletal+muscle+injury.">Numb is required to prevent p53-dependent senescence following skeletal muscle injury.</a> Nat Commun. 6, 8528 (2015).</li><li>Debacq-Chainiaux, F., Erusalimsky, J. D., Campisi, J., Toussaint, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+to+detect+senescence-associated+beta-galactosidase+(SA-betagal)+activity,+a+biomarker+of+senescent+cells+in+culture+and+in+vivo.">Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo.</a> Nat Protoc. 4 (12), 1798-1806 (2009).</li><li>Dimri, G. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biomarker+that+identifies+senescent+human+cells+in+culture+and+in+aging+skin+in+vivo.">A biomarker that identifies senescent human cells in culture and in aging skin in vivo.</a> Proc Natl Acad Sci U S A. 92 (20), 9363-9367 (1995).</li><li>Lee, B. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescence-associated+beta-galactosidase+is+lysosomal+beta-galactosidase.">Senescence-associated beta-galactosidase is lysosomal beta-galactosidase.</a> Aging Cell. 5 (2), 187-195 (2006).</li><li>Krishna, D. R., Sperker, B., Fritz, P., Klotz, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+pH+6+beta-galactosidase+activity+indicate+cell+senescence?.">Does pH 6 beta-galactosidase activity indicate cell senescence?.</a> Mech Ageing Dev. 109 (2), 113-123 (1999).</li><li>Cristofalo, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SA+beta+Gal+staining:+biomarker+or+delusion.">SA beta Gal staining: biomarker or delusion.</a> Exp Gerontol. 40 (10), 836-838 (2005).</li></ol>

Burada, senescent her ikisi de algılamak için bir yöntem ve pluripotent kök hücre içinde Bilişim farelerin kas iskelet mevcut. Bu yöntem değerlendirmek ve her iki yaşlanma ölçmek ve hücresel plastisite vivo içindeikna etmek ve yaşlanma doku onarımı ve rejenerasyon rol incelemek için kullanılabilir.
Geçerli protokol, yaşlanma ilişkili β-galaktozidaz (SA-β-Gal) tahlil içinde vivo senescent hücreleri kas iskelet algılamak için kullanılır. Bu tahlil ...

Hücresel yaşlanma stres yanıt istikrarlı hücre döngüsü tutuklama tarafından karakterize bir şeklidir. Son on yıl içinde araştırma sıkıca yaşlanma embriyonik geliştirme, fibrozis ve1,2yaşlanma organizma da dahil olmak üzere çeşitli biyolojik ve patolojik işlemlerle ilişkili olduğunu kurmuştur. Hücresel yaşlanma ilk3kısalma telomer tarafından tetiklenen ikileştirici onların ömrü sonundaki insan fibroblast tespit edilmiştir. İkileştirici stres yanı sıra, yaşlanma, DNA hasarı, oksidatif stres, oncogenic sinyalleri ve genomik/epigenomic değişiklikler, herhangi biri sonunda p53/p21 ve/veya pRB yolları etkinleştirebilir gibi neden olabilir birçok çekim gücü vardır kurmak ve kalıcı büyüme tutuklama1güçlendirmek. Senescent hücreleri önemli özelliklerinden biri olan metabolik olarak aktif kalır ve sağlam bir yaşlanma ilişkili salgı fenotip (SASP) hızlı: salgısı birçok inflamatuar sitokinlerin, büyüme faktörleri ve hücre dışı matriks faktör4. SASP faktörler arabuluculuk ve bağışıklık hücreleri çeken ve yerel ve sistemik doku ni1değiştiren güçlü etkileri nedeniyle yaşlanma etkisi yükseltecek önemli bir rol oynamak için önerilmiştir. İlginçtir, yaşlanma son zamanlarda doku onarımı ve yenilenme için5,6önemli olmak teklif edildi. Buna ek olarak, veri--dan bizim de dahil olmak üzere birkaç labs doku zarar indüklenen yaşlanma SASPs yenilenme7-9tanıtmak için üzerinden hücresel plastisite geliştirmek önerdi. Bu nedenle, tüm gelişmekte olan verileri yaşlanma içinde vivoeğitim önemini vurgulayın.
Yazı İndüklenmiş pluripotent kök hücre (IPSC) dönemde, hücresel plastisite bir hücrenin yeni bir kimlik edinme ve farklı uyaranların her iki kültür ve in vivo10maruz alternatif bir kader benimsemeye kapasitesidir. Tam yeniden programlama elde vivo içinde11,12, olabilir bilindiği nerede ifade dört Yamanaka faktör içeren kaset: Oct4, Sox2, Klf4ve c-Myc (OSKM) indüklenen vivo içinde birden fazla organ teratomas oluşumu teşvik olabilir. Bu nedenle, bir etiketteki fare modeli (i4F) güçlü bir sistemi kritik düzenleyiciler ve hücresel plastisite11için önemli yollar tanımlamak için kullanılabilir.
Uygun ve hassas vivo içinde sistem nasıl hücresel yaşlanma anlamak önemlidir doku rejenerasyonu bağlamında hücresel plastisite düzenler. Burada, sağlam bir sistem ve yaşlanma ve doku rejenerasyonu bağlamında hücresel plastisite arasındaki bağlantıyı değerlendirmek için detaylı bir iletişim kuralı mevcut. Cardiotoxin (CTX) indüklenen kombinasyonu kas hasarı Tibialis Anterior (TA) kas grubunda, doku rejenerasyonu ve i4F fare modeli çalışmaya iyi kurulmuş bir sistem hücresel yaşlanma ve içinde vivo algılanmasını sağlar kas yeniden oluşturma işlemi sırasında yeniden programlama.
Hücresel plastisite ile yaşlanma arasında bağlantı değerlendirmek için i4F fare CTX ile akut kas hasarı ikna etmek için yaralı ve in vivo yeniden programlama ikna etmek için Doksisiklin ile (0.2 mg/mL) 7 gün boyunca tedavi. Bir CTX akut kas hasarı indüklenen ve rejenerasyon Protokolü son yayınlanan13, etik nedenlerle oldu iken, bu yordamı geçerli iletişim kuralında atlanacak. Senescent hücreleri zirvesine daha önce gözlemlenmiştir ne zaman14TA kas örnekleri 10 gün sonrası yaralanma13' toplanacak. Burada, bu ayrıntılı protokolünü yaşlanma (yolu ile SA-β-Gal) düzeyini ve programlama (via IHC MicroRNAs boyama) değerlendirmek için gerekli olan adımları açıklar.
Yaşlanma ile ilişkili beta-galaktozidaz (SA-β-Gal) tahlil senescent hücreler kültür ve in vivo15içinde algılamak için en sık kullanılan tahlil olduğunu. Diğer deneyleri için karşılaştırıldığında, SA-β-Gal tahlil için in vivo çalışmada özellikle önemlidir sağlam doku mimarisi ile yerel onların ortamında senescent hücreleri tanımlaması sağlar. Ayrıca, SA-β-Gal tahlil IHC kullanarak diğer işaretleri ile çift mümkündür. Ancak, SA-β-Gal tahlil örnekleri, taze veya dondurulmuş olan büyük bir sınırlama kalır gerektirir. Donmuş TA kas numuneler gibi taze veya dondurulmuş dokular düzenli olarak kullanılabilir olduğunda, SA-β-Gal belli ki senescent hücreleri algılamak için en uygun tahlil olduğunu. MicroRNAs olduğunu reprogramed hücreleri iki nedenden dolayı algılamak için kullanılan işaretleyici: 1) pluripotency için; temel bir işaretleyicidir 2) daha da önemlisi, onun ifade Doksisiklin (dox) tarafından tahrik değil, bu nedenle Yamanaka kaset zorla ifade yerine İndüklenmiş Pluripotent algılar.
Bu çalışmada sunulan boyama iletişim kuralları ayrı ayrı miktar yordamı kolaylaştırmak için yapılan, ama da senescent her ikisi de görselleştirmek için ortak boyama bir yordam ve pluripotent kök hücreler aynı bölümünde yapılabilir unutmamak önemlidir.

<table>
	<tbody>
		<tr>
			<td>K3Fe(CN)6</td>
			<td>Sigma</td>
			<td>13746-66-2</td>
			<td>For SA-&#946; Gal staining solution</td>
		</tr>
		<tr>
			<td>K4Fe(CN)6</td>
			<td>Sigma</td>
			<td>14459-95-1</td>
			<td>For SA-&#946; Gal staining solution</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>7786-30-3</td>
			<td>For SA-&#946; Gal staining solution</td>
		</tr>
		<tr>
			<td>X-Gal</td>
			<td>Sigma</td>
			<td>B4252</td>
			<td>For SA-&#946; Gal staining solution</td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Sigma</td>
			<td>D3447</td>
			<td>For inducing in vivo reprogramming</td>
		</tr>
		<tr>
			<td>Cardiotoxin</td>
			<td>Lotaxan Valence, France</td>
			<td>L8102</td>
			<td>For muscle injury</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma</td>
			<td>111-30-8</td>
			<td>For Fixation solution</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron microscopy science</td>
			<td>50-980-487</td>
			<td>For Fixation solution</td>
		</tr>
		<tr>
			<td>NaCitrate : Sodium Citrate monobasic bioxtra, anhydre</td>
			<td>Sigma</td>
			<td>18996-35-5</td>
			<td>For permeabilization solution</td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>Sigma</td>
			<td>93443</td>
			<td>For permeabilization solution</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A3608</td>
			<td>Washing solution</td>
		</tr>
		<tr>
			<td>Antibody anti- Nanog</td>
			<td>Cell signalling</td>
			<td>8822S</td>
			<td>Rabbit monoclonal antibody</td>
		</tr>
		<tr>
			<td>EnVision+ Kits (HRP. Rabbit. DAB+)</td>
			<td>Dako</td>
			<td>K4010</td>
			<td>For Nanog revelation</td>
		</tr>
		<tr>
			<td>Eosin 1%</td>
			<td>Leica</td>
			<td>380159EOF</td>
			<td>Counterstainning</td>
		</tr>
		<tr>
			<td>Fast red</td>
			<td>Vector Laboratories</td>
			<td>H-3403</td>
			<td>Counterstainning</td>
		</tr>
		<tr>
			<td>Thermo Scientific Shandon Immu-Mount</td>
			<td>Fisher scientific</td>
			<td>9990402</td>
			<td>Mounting solution</td>
		</tr>
		<tr>
			<td>Quick-hardening mounting medium for microscopy : Eukitt&#174;</td>
			<td>Sigma</td>
			<td>25608-33-7</td>
			<td>Mounting solution</td>
		</tr>
		<tr>
			<td>Microscope Phase Contrast Brightfield CKX41: 10X-20X-40X objectives</td>
			<td>Olympus</td>
			<td>CKX41</td>
			<td>Microscope for Nanog quantification</td>
		</tr>
		<tr>
			<td>Mouse: i4F-A</td>
			<td>Abad et al., 2013</td>
			<td>N/A</td>
			<td>Reprogrammable mouse model</td>
		</tr>
		<tr>
			<td>Skeletal muscle, Tibialis Anterior</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Scanner</td>
			<td>Zeiss</td>
			<td>Axio Scan Z1</td>
			<td>slides scanning</td>
		</tr>
	</tbody>
</table>

evaluation of injury-induced senescence and in vivo reprogramming in the skeletal muscle

Hücresel yaşlanma kanser ve yaşlanma gibi birçok fizyolojik ve patolojik süreçler için önemlidir istikrarlı hücresel büyüme krizi ile karakterize bir stres yanıttır. Son zamanlarda, yaşlanma Ayrıca doku onarımı ve rejenerasyon karıştığı olmuştur. Bu nedenle, senescent hücreleri vivotanımlamak için giderek kritik haline gelmiştir. Yaşlanma ile ilişkili β-galaktozidaz (SA-β-Gal) tahlil senescent hücreleri hem de kültür ve in vivoalgılamak için en çok kullanılan tahlil olduğunu. Bu tahlil senescent hücreleri artan lizozomal içeriğinde lizozomal β-galaktozidaz faaliyet histochemical algılanması suboptimum pH (6 veya 5.5) sağlayan temel alır. Akış Sitometresi gibi diğer deneyleri ile karşılaştırıldığında bu doku mimari, Morfoloji, ilgili konumu gibi değerli bilgiler sunan ikamet çevreleri senescent hücrelerde tanımlaması sağlar ve Kaplin immünhistokimya (IHC) üzerinden diğer imleçli olasılığı. Büyük SA-β-Gal tahlil taze veya dondurulmuş örnekler şartı kısıtlamasıdır.
Burada, nasıl hücresel yaşlanma anlamak için detaylı bir iletişim kuralı mevcut hücresel plastisite ve doku rejenerasyonu içinde vivoteşvik etmektedir. SA-β-Gal senescent hücreleri iskelet kas doku rejenerasyonu çalışmaya köklü bir sistemdir yaralanma üzerine algılamak için kullanır. Ayrıca, IHC MicroRNAs, bir işaretleyici pluripotent kök hücrelerin bir transgenik fare modelinde algılamak için kullanır. Bu iletişim kuralı incelemek ve hücresel yaşlanma indüklenen hücresel plastisite ve in vivo yeniden programlama bağlamında ölçmek sağlar.

Kas yaralanması indüklenen hücresel yaşlanma algılama
Son zamanlarda kas yaralanması geçici hücresel yaşlanma14indükler kanıtlanmıştır. 10 gün sonrası yaralanma (DPI), bozuk myofibers çoğunluğu yenilenme merkezi konuma sahip çekirdeklerin, myofibers, yenileyici bir hallmark ile geçiren ve kas mimarisini yeniden kurulur. İnfiltrating inflamatuar hücrelerin belirli bölgelerde görünür...

Watch this Scientific Journal Video about Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle at JoVE.com

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Yaralanma kaynaklı yaşlanma ve In Vivo kas iskelet yeniden programlama değerlendirilmesi

Burada senescent her ikisi de algılamak için detaylı bir protokol ve pluripotent kök hücre iskelet kas üzerine vivo içinde yeniden programlama inducing sırasında yaralanma mevcut. Bu yöntem doku yeniden oluşturma işlemi sırasında hücresel yaşlanma rolünün değerlendirilmesi ve in vivoyeniden programlama için uygundur.

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

The overall goal of this protocol is to evaluate both in vivo senescence and reprogramming in the skeletal muscle upon tissue injury. This method can help to address critical questions in the regenerative medicine field, such as how to enhance cellular plasticity to promote muscle regeneration. The main advantage of this technique is to provide a reliable method to detect and quantify senescence cells in the resident environment.Cell senescence has been recently implicated in tissue repair and regeneration. Understanding the mechanisms of how senescence contributes to tissue repair and the regeneration will certainly have an impact on regenerative medicine. We first had the idea for this method when we investigated the potential impact of tissue damage on in vivo reprogramming in skeletal muscle.To begin this procedure, fix the slides of muscle samples in 1%paraformaldehyde and 0.2%glutaraldehyde in PBS for four minutes. Then, wash the slides with PBS two times, each time for 10 minutes. Then, wash the slides with PBS, pH 5.5 for 30 minutes.Next, incubate the sections with the X-gal solution in the dark at 37 degrees Celsius for a minimum of 24 hours. Afterward, wash the slides with PBS three times, each time for 10 minutes. Then, post-fix the slides with PBS containing 1%paraformaldehyde for 30 minutes.Finally, wash the slides with PBS three times. Subsequently, counterstain with 0.2%eosin by immersing the slides in the eosin solution for one minute. Then, rinse them with distilled water briefly.Mount the slides with aqueous non-fluorescing mounting medium. Fix the slides with PBS containing 4%paraformaldehyde for 10 minutes. Wash them with PBS twice, each time for 10 minutes.Subsequently, add the permeabilization solution, and incubate for five minutes. Following this, wash the slides with PBS twice, each time for five minutes. In the last wash, add 200 microliters of PBS, containing 0.25%BSA, directly on the slides for five minutes.Incubate the slides with the primary anti-Nanog antibody at 1.25 micrograms per milliliter overnight at 4 degrees Celsius in PBS containing 5%FBS. Wash the slides with PBS twice, each time for 10 minutes. In the last wash, use 200 microliters of PBS containing 0.25%BSA for five minutes.Subsequently, incubate the slides with 100 microliters of RAB-HRP secondary antibody from a ready-to-use kit for 45 minutes. Then, wash the slides with PBS three times, each time for five minutes to remove the secondary antibody. After that, dilute DAB in the substrate buffer, provided by the ready-to-use kit.Vortex vigorously. Add 100 microliters of DAB solution, directly onto each slide, and incubate for a maximum of 10 minutes at room temperature. Next, remove DAB solution by rinsing the slides with water.Counterstain the slides with fast red solution for two minutes. Then, wash the slides with water again, briefly. Following that, dehydrate them with 95%ethanol for five minutes, followed by 100%ethanol twice, each time for five minutes.After that, mount the slides with quick-hardening mounting medium. Observe the slides under the microscope in brightfield at 20x to avoid background. In this procedure, scan the slides and choose the two best quality sections on each slide with the maximum possible distance in-between base on tissue integrity, quality of the staining, and counterstaining.Count the SA-Beta-Gal positive cells in these two sections and calculate the mean value. Then, determine the rank of the pixel size by manually selecting the smallest and the largest positive SA-Beta-Gal cells. In ImageJ Interface, click Analyze, Tools, ROI Manager, Analyze, Set Measurement.Then, select Area. Use the selection tool and surround the smallest and biggest positive SA-Beta-Gal cell, and add it to the ROI Manager by clicking on, Add. After that, measure the sizes, using the Measure button, and save the values for later use.Next, adjust the threshold parameter to ensure all the visible positive cells are selected. Convert the image to grayscale by clicking, Image, Type, then 8-bit. Afterward, go to Image, Adjust, and Threshold.Move the second cursor until all the positive SA-Beta-Gal cells are covered in red, then click, Apply. Analyze the particles by clicking, Analyze, Analyze Particles, followed by Size and apply the value defined earlier. Enter Circularity, 0.00-1.00, and click OK.A summary of all the counted particles will be shown in the ROI Manager.Now, transfer all the selected particles to the original image. Adjust the selection manually to ensure accurate quantification. Add positive cells or remove false positive cells.Finally, measure the area by outlining the section using the selection tool. Click ROI Manager, Add, then Measure. Under the microscope, count the Nanog positive cells in the brightfield manually at 20x magnification.Shown here is a schematic representation to evaluate in vivo reprogramming and senescence level after muscle injury. Here are the representative pictures of SA-Beta-Gal and Nanog staining on the frozen sections of damaged skeletal muscle. These images show SA-Beta-Gal staining, counterstained with eosin, and the staining of Nanog, counterstained with fast red.The non-injured muscles are shown on the top, whereas the injured muscles are shown at the bottom. This graph shows the quantification and correlation of SA-Beta-Gal positive and Nanog positive cells in consecutive sections at 10 days post-injury. While attempting this procedure, it's important to remember to always include a negative control to ensure the specificity of the staining.Following this procedure, all the methods, like immunofluorescence, choosing different marker of senescence, all cell types can be performed in order to answer additional questions, like the cellular identity of the senescent cells. After each development, this technique paved the way for researchers in the field of senescence, or tissue repair and regeneration, to explore how cellular senescence can promote cellular plasticity to facilitate tissue repair and regeneration in mammals. So after watching this video, you should have a good understanding of how to identify both senescent and reprogrammed cells in vivo.

evaluation-injury-induced-senescence-vivo-reprogramming-skeletal

Research

JoVE Journal

Developmental Biology

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Evans Blue Dye ile Zebra balığı Larva İskelet Kası Bütünlüğü Analizi

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Gelişim Biyolojisi

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 ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Cardiotoxin Enjeksiyonu ile Akut İskelet Kası Regeneration indüksiyon

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Diseksiyon ve mantar vücut immünfloresan boyama ve yetişkin Drosophila melanogaster beynindeki nöronlar Photoreceptor

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

Contractile fare faaliyet kaynaklı kas iskelet fenotipik uyarlamalar çalışmaya kronik stimulasyon uygulanması

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Kas iskelet uydu hücreleri tarafından Pax7 ve Laminin antikorlar ile ayirt tanımlaması

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

Vivo Kas-tendon morfogenez Drosophila pupa içinde görüntüleme

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

İndüksiyon ve birincil insan hücrelerinde hücresel yaşlanma doğrulama

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. Recent studies have extended the role of cellular senescence to various physiological processes, including development, wound healing, immune surveillance, and age-related tissue dysfunction. Although cell cycle arrest is a critical hallmark of cellular senescence, an increased intracellular reactive oxygen species (ROS) production has also been demonstrated to play an important role in the induction of cellular senescence. In addition, recent studies revealed that senescent cells exhibit potent paracrine activities on neighboring cells and tissues through a senescence-associated secretory phenotype (SASP). The sharp increase in interest regarding therapeutic strategies against cellular senescence emphasizes the need for a precise understanding of senescence mechanisms, including intracellular ROS and the SASP. Here, we describe protocols for quantitatively assessing intracellular ROS levels during H-Ras-induced cellular senescence using ROS-sensitive fluorescent dye and flow cytometry. In addition, we introduce sensitive techniques for the analysis of the induction of mRNA expression and secretion of SASP factors. These protocols can be applied to various cellular senescence models.

Intracellular ROS has been shown to play an important role in the induction of cellular senescence. Here, we describe a sensitive assay for quantifying ROS levels during cellular senescence. We also provide protocols for assessing the senescence-associated secretory phenotype, which reportedly contributes to various age-related dysfunctions.

Reaktif oksijen türleri ve yaşlanma ilişkili salgı fenotip Normal insan fibroblast içinde bir nicel ölçüm onkogen kaynaklı yaşlanma sırasında

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.