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

우리는 Clemire Cimper에 그녀의 우수한 기술 지원에 대 한 빚을입니다. H.L.의 연구실에서 작업 파스퇴르, 검색 과학 센터 국가 부 라와 직원 회 드 라 검색 (응용 d'Excellence, 소생 Investissement d'Avenir;에 의해 투자 되었다 ANR-10-LABX-73), 직원 회 드 라 검색 (ANR-16-CE13-0017-01) 및 Fondation 호 (PJA 20161205028). 참조 및 교류는 박사에 의해 부활 컨소시엄에서 박사 장학 자금을.

동물 인 파스퇴르 (CETEA) 승인 프로토콜의 윤리 위원회와 유럽 공동체 지침에 의하여 처리 했다.
 1. 재고 솔루션의 준비 <ol><li>근육 샘플 고정을 위한 자료 준비. 20 mL 물 근육 고정을 위한 동결 포함 매체 수 있도록 RT에서 tragacanth 껌의 0.5 g을 녹.</li>
	<li>SA-β-Gal 얼룩에 대 한 솔루션을 준비.
	<ol><li>재고의 솔루션 K 3 Fe(CN) 6 (100 m m), K 4 Fe(CN) ...

<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>

여기, 선물이 모두 노화를 검출 하는 방법 및 pluripotent 줄기 세포 재프로그래밍 쥐의 골격 근육. 이 방법은 평가 척도 모두 노화 세포가 소성에 vivo에서, 유도 그리고 조직 수리 및 재생에 노화의 역할을 검토 사용 될 수 있습니다.
현재 프로토콜 골격 근육에 노화 세포 vivo에서 감지 하 노화 관련 된 β-galactosidase (SA-β-Gal) 분석 결과 사용 합니다. 이 분석 결과 중형 p...

세포 노화 스트레스 응답 안정적인 세포 주기 검거가 특징의 한 형태입니다. 지난 10 년간에서 연구는 단단히 설립 노화 배아 개발, 섬유 증, 및1,2노화 하는 유기 체를 포함 하 여 다양 한 생물 학적 및 병 적인 프로세스와 연결 된. 세포 노화는 그들의 일차 수명 telomere 단축3에 의해 실행의 끝에 인간의 섬유 아 세포에서 처음 확인 되었다. 일차 스트레스, 노화, DNA 손상, 산화 스트레스, 종양 신호는의 결국에 p53/p21 또는 pRB 통로 활성화 하는 게놈/epigenomic 변경 등을 일으킬 수 있는 다른 많은 자극 있습니다. 설정 하 고 영구 성장 체포1강화. 노화 세포의 중요 한 특성 중 하나는 그들은 metabolically 활성 상태로 유지 하 고 견고 하 게 표현 하는 노화에 관련 된 분 비 형 (SASP): 많은 염증 성 cytokines, 성장 인자, 세포 외 매트릭스의 분 비 요인4. SASP 요소를 중재 하 고 면역 세포를 유치 하 고 지역 및 조직의 조직 milieus1변경에 그들의 강력한 효과 인해 노화 효과 증폭에 중요 한 역할을 제안 되었습니다. 흥미롭게도, 노화 최근 제안 되었습니다 조직 수리 및 재생5,6에 대 한 중요 한 것. 또한, 우리를 포함 한 여러 연구소에서 데이터 조직 손상-유도 노화 세포가 소성, SASPs, 재생7-9홍보를 통해 강화 수 있습니다 제안 했다. 따라서, 모든 신흥 데이터 노화 비보의 중요성을 강조 표시 합니다.
게시물 유도 만능 줄기 세포 (iPSC) 시대에 세포가 소성은 새로운 정체성을 획득 하 고 두 문화 그리고 vivo에서10에 다른 자극에 노출 되 면 대체 운명 채택 셀의 능력 이다. 그것은 알려져 그 전체 프로그래밍 하 달성 vivo에서11,12, 수 어디의 표현은 4 야 마나카 요소를 포함 하는 카세트: Oct4, Sox2, Klf4및 c-Myc (OSKM) 유도 수 비보에 여러 장기에 teratomas 형성을 촉진. 따라서, 중요 한 레 귤 레이 터와 셀룰러 소성11에 대 한 중요 한 경로 식별 하는 강력한 시스템으로 재프로그래밍 마우스 모델 (i4F)를 사용할 수 있습니다.
적합 하 고 민감한 vivo에서 시스템은 어떻게 세포 노화를 이해에 필수적인 조직 재생의 맥락에서 세포가 소성을 조절. 여기, 우리는 강력한 시스템 및 노화와 조직 재생의 맥락에서 셀룰러 소성 사이의 링크를 평가 하는 상세한 프로토콜 제시. 유도 하는 cardiotoxin (CTX)의 조합 근육 손상 Tibialis 앞쪽 (TA) 근육 그룹, 조직 재생, i4F 마우스 모델을 공부 하 고 잘 설립 시스템에에서 수 세포 노화와 생체 내에서 의 검색 근육 재생성 하는 동안 프로그래밍.
세포가 소성 및 노화 사이의 링크를 평가 하려면 i4F 마우스 급성 근육 손상을 유발 하는 CTX 부상 되며 doxycycline (0.2 mg/mL) 7 일 이상 vivo에서 재프로그래밍을 유도 하는 것으로 치료. CTX 급성 근육 손상을 유발 하는 동안 재생 프로토콜 윤리적인 이유를 위해 최근에 출판 된13되었습니다 현재 프로토콜에이 절차를 생략 됩니다. 노화 세포의 피크 이전 관찰 되었습니다14때 따 근육 샘플 10 일 게시물 부상13, 수집 됩니다. 여기,이 상세한 프로토콜 (SA-β-Gal)를 통해 노화의 수준 및 재활 (IHC Nanog의 얼룩)를 통해 평가 하는 데 필요한 모든 단계를 설명 합니다.
노화 관련 베타-galactosidase (SA-β-Gal) 분석 결과 문화 그리고 vivo에서15에 노화 세포 모두를 감지 하는 가장 일반적으로 사용 되 분석 결과가 이다. 다른 분석 실험에 비해, SA-β-Gal 분석 결과 그대로 조직 아키텍처, vivo에서 학문에 대 한 특히 중요 한 그들의 네이티브 환경에서 노화 세포의 식별 수 있습니다. 또한, IHC를 사용 하 여 다른 표식으로 SA-β-Gal 분석 결과 하 가능 하다. 그러나, SA-β-Gal 분석 결과 주요 제한 남아 있는 신선 또는 냉동 샘플 필요지 않습니다. 신선 또는 냉동 조직 냉동된 따 근육 샘플 같은 정기적으로 사용할 수 있을 때, SA-β-Gal은 명백히 노화 세포를 감지 하는 가장 적합 한 분석 결과 이다. Nanog는 두 가지 이유로 reprogramed 셀을 검출 하는 데 사용 하는 마커: 1) 그것은 pluripotency;에 대 한 필수적인 마커 2) 더 중요 한 것은, 그 표현 doxycycline (dox)에 의해 구동 하지 않으면, 따라서 유도 pluripotency 보다는 야 마나카 카세트의 강제 식 감지.
그것은 중요 한 것,이 연구에서 제시 하는 얼룩 프로토콜 정량화 절차를 단순화 하기 위해 별도로 실시 될 수 있지만 모두 노화를 시각화 하는 공동 얼룩 절차 및 pluripotent 줄기 세포 동일한 섹션에서 수행할 수 있습니다.

<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

세포 노화는 안정적인 세포 성장 체포, 암과 노화 등 많은 생리와 병리학 과정에 대 한 중요 한 특징은 스트레스 반응이 다. 최근, 노화 또한 조직 수리 및 재생에 연루 되었습니다. 따라서, 그것은 점점 노화 세포에 vivo에서식별에 중요 한 되고있다. 노화 관련 된 β-galactosidase (SA-β-Gal) 분석 결과 노화 세포 모두 문화에 vivo에서감지 하는 가장 널리 사용 되 분석 결과가 이다. 이 분석 결과 기반 노화 세포에서 증가 lysosomal 내용을 lysosomal β-galactosidase 활동의 조직화 학적인 탐지 중형 pH 6 (5.5)에서 수 있습니다. 다른 분석 실험, cytometry, 같은 비교 관련 조직 구조, 형태, 위치와 같은 중요 한 정보를 제공 합니다 그들의 거주 환경에 노화 세포의 식별 수 있습니다 그리고 커플링 immunohistochemistry (IHC)를 통해 다른 마커와 가능성. SA-β-Gal 분석 결과의 주요 한계는 신선 또는 냉동 샘플의 요구 사항입니다.
여기, 우리가 어떻게 세포 노화를 이해 하는 상세한 프로토콜 현재 셀룰러 소성 및 조직 재생 비보를 촉진. SA-β-Gal 사용 하 여 조직 재생 연구에 기초가 튼튼한 시스템은 부상 시 골격 근육에 노화 세포를 감지. 또한, IHC 사용 하 여 유전자 변형 마우스 모델에서 Nanog, 만능 줄기 세포의 마커를 감지. 이 프로토콜 검사 하 고 세포 노화 유발된 세포가 소성 그리고 vivo에서 프로그래밍의 맥락에서 정할 수 있습니다.

근육 부상 유발 세포 노화를 감지
그것은 최근에 입증 되었습니다 근육 부상을 과도 세포 노화14유도. 10 일 후 부상 (DPI)에서 손상 된 myofibers의 대부분 위치 핵, myofibers, 재생의 품질 증명으로 재생을 겪고 있다 그리고 근육의 아키텍처는 다시 설립. 침투 염증 세포는 특정 지역에 표시 하면서 극적으로 감소 된다...

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

부상-유도 노화 및 골격 근육에서 프로그래밍을 Vivo에서 의 평가

여기 선물이 모두 노화를 감지 하는 상세한 프로토콜 및 만능 줄기 세포 유도 vivo에서 프로그래밍 하는 동안 부상에 골격 근육. 이 메서드는 조직 재생성 하는 동안 세포 노화의 역할을 평가 하 고 vivo에서프로그래밍에 적합 합니다.

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.

에반스 블루 염료와 Zebrafish의 애벌레 골격 근육 무결성 분석

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

연구

발생학

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.

높은 처리량 마이크로 호흡 측정을위한 마우스 골격근의 최소 수량에서 미토콘드리아 격리 사용

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 주입에 의한 급성 골격 근육 재생 유도

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.

해 부 및 Immunofluorescent 얼룩의 버섯 몸과 성인 초파리 melanogaster 두뇌 신경 세포 포토 리셉터

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.

수축 성 활동 유도 된 쥐 골격 근육 Phenotypic 적응 연구 만성 자극에의 응용

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.

Pax7 Laminin 항 체와 면역 형광에 의해 골격 근육 인공위성 세포의 식별

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.

Vivo에서 근육-힘 줄 Morphogenesis 초파리 번데기에의 이미징

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.

유도 및 기본 인간 세포에서 세포 노화의 유효성 검사

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.

Oncogene 유도 노화 중 반응성 산소 종 및 정상적인 인간 섬유 아 세포에서 분 비 형 노화 관련의 정량적 측정

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.