저자는이 프로토콜의 촬영 기간 동안 C. Zambataro의 기술 지원을 인정하고 싶습니다. 우리는 기꺼이 글렌 네이선 충격 센터 상을 의학 연구 (SM), Hillblom 재단과 국립 보건원에 대한 재단 (P30AG025708)와 PO1AG025901의 지원을 인정합니다. JMF는 노화 연구에 대한 책임 연구원에게 수여 T32AG000266에 의해 지원되었다.

<ol>
	<li>Wolska, B. M., Solaro, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+isolation+of+adult+mouse+cardiac+myocytes+for+studies+of+contraction+and+microfluorimetry.">Method for isolation of adult mouse cardiac myocytes for studies of contraction and microfluorimetry.</a> Am. J. Physiol. 271 (3 Pt 2), H1250-H1255 (1996).</li><li>Shioya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+isolating+healthy+heart+cells+from+mouse+models.">A simple technique for isolating healthy heart cells from mouse models.</a> J. Physiol. Sci. 57 (6), 327-335 (2007).</li><li>Guo, G., Huss, M., Tong, G. Q., Wang, C., Li Sun, L., Clarke, N. D., Robson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+cell+fate+decisions+revealed+by+single-cell+gene+expression+analysis+from+zygote+to+blastocyst.">Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst.</a> Dev. Cell. 18 (4), 675-685 (2010).</li><li>Narsinh, K. H., Sun, N., Sanchez-Freire, V., Lee, A. S., Almeida, P., Hu, S., Jan, T., Wilson, K. D., Leong, D., Rosenberg, J., Yao, M., Robbins, R. C., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+transcriptional+profiling+reveals+heterogeneity+of+human+induced+pluripotent+stem+cells.">Single cell transcriptional profiling reveals heterogeneity of human induced pluripotent stem cells.</a> J. Clin. Invest. 121 (3), 1217-1221 (2011).</li><li>D'haene, B., Vandesompele, J., Hellemans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+and+objective+copy+number+profiling+using+real-time+quantitative+PCR.">Accurate and objective copy number profiling using real-time quantitative PCR.</a> Methods. 50 (4), 262-270 (2010).</li><li>Cook, M. 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+Anatomy+of+the+Laboratory+Mouse.">The Anatomy of the Laboratory Mouse.</a> Informatics. , (1965).</li><li>Gaillard, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aorta.">Aorta.</a> Radiopaedia. , (2008).</li><li>St&#229;hlberg, A., Andersson, D., Aurelius, J., Faiz, M., Pekna, M., Kubista, M., Pekny, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+cell+populations+with+single-cell+gene+expression+profiling:+correlations+and+identification+of+astrocyte+subpopulations.">Defining cell populations with single-cell gene expression profiling: correlations and identification of astrocyte subpopulations.</a> Nucleic Acids Res. 39 (4), e24-e24 (2011).</li><li>Zhong, J. F., Chen, Y., Marcus, J. S., Scherer, A., Quake, S. R., Taylor, C. R., Weiner, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+processor+for+gene+expression+profiling+of+single+human+embryonic+stem+cells.">A microfluidic processor for gene expression profiling of single human embryonic stem cells.</a> Lab Chip. 8 (1), 68-74 (2008).</li><li>Bahar, R., Hartmann, C. H., Rodriguez, K. A., Denny, A. D., Busuttil, R. A., Dollé, M. E., Calder, R. B., Chisholm, G. B., Pollock, B. H., Klein, C. A., Vijg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+cell-to-cell+variation+in+gene+expression+in+ageing+mouse+heart.">Increased cell-to-cell variation in gene expression in ageing mouse heart.</a> Nature. 441 (7096), 1011-1014 (2006).</li><li>Raj, A., van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature,+nurture,+or+chance:+stochastic+gene+expression+and+its+consequences.">Nature, nurture, or chance: stochastic gene expression and its consequences.</a> Cell. 135 (2), 216-226 (2008).</li><li>Elowitz, M., Levine, A., Siggia, E., Swain, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+gene+expression+in+a+single+cell.">Stochastic gene expression in a single cell.</a> Science. 297 (5584), 1183-1186 (2002).</li></ol>

<p class="jove_content"이 문서의&gt; 무료 접속이 생산은 시그마 - 올드 리치를 후원합니다.</p

이 메서드는 기능의 숫자뿐만 아니라 유전자 발현 연구에 대한 cardiomyocytes를 생성할 수있는 가능성이 있습니다. 단일 세포의 검사는 유전자 발현 분석에 급성장 지역입니다. 단일 세포 수준에서 transcriptional 수준을 검토의 장점은 전체 조직의 준비에서 가능하지 않은 순수한 세포 인구의 시험을 허용한다는 것입니다. 또한, 단일 세포 분석은 이전에 균질 8,9 것으로 생각되었다 세포 인구를 정의하는 각각의 세포에서 mRNA 수준의 확률적 변화의 시험을 수 있습니다. 자신의 유전자 표현 10,11,12에 잠재적으로 확률적 아르 유전자를 식별하는 이외에 방법은 또한 유전자 발현 프로파일에 의해 정의된 희귀 세포 집단의 식별을 허용합니다. 이 방법은 유전자 발현 분석에 흥미로운 가능성이 사용하는 다수의를 제공하고 있지만, som있다단일 세포의 사용에 전자주의 사항 및 고려 사항. 단일 세포 분석에 기본 제한은, 예를 들어 분산에 대한 관심의 메트 릭에 관련하여 통계적 중요성을 달성하기 위해 실험에서 충분한 세포의 모음입니다. 관심의 조직으로부터 격리 세포의 숫자가 제한되지 않습니다 경우에, 하나는 같은 차세대 시퀀싱, 또는 nanofluidic 배열과 같은 접근법을 사용하여 그룹 당 세포의 수백을 확인하실 수 있습니다. 하지만 일부 경우에, 관심의 조직에서 충분한 세포를 얻기에 어려움이있을 수 있습니다. 이 부분에서 대상 셀 형식의 컬렉션에 대한 특이한 시간 집중 과정으로 인해 발생할 수 있습니다. 그러나, 단일 셀 컬렉션을 진행하기 위해 신중 계획과 사전 준비는 여전히 제한된 기술 오류와 함​​께 엄격한 단일 세포 분석을 위해 허용할 수 있습니다. 그것이 고려되어야 그 결과를 검토하면, 세포 분리에 대해이 절차는 여러 studie에 사용되었습니다 비록 그s이 (현미경, 전기 생리학, 등), 격리 자체는 잠재적으로 유전자 발현에 영향을 수 있습니다. 생물 학적 샘플을 조작하는 방법과 마찬가지로, 당신의 결과의 결과를주의 깊게 단세포 표현은 조직 자체 기술하지 편견 대표하기 위해 검증해야합니다. 이러한 현장 하이브 리다이 제이션에서와 같이 방법은 손상 조직에서 이러한 결과를 확인하기 위해 유용합니다. 마지막으로, 그것은 생성된 데이터가 신중하게 품질 관리를위한 체크되어 있는지 확인하는 것이 중요합니다. 그림 8에 표시된 데이터는 qPCR의 assays 이러한 분석 # 13 (A13)와 같은, 그들의 특이성이 매우 강력한하거나 그러한 분석 # 34 (A34)와 같은 기술적인 변화로 이어질 수있는 다양성의 높은 수준을 가지고 수 있습니다 보여줍니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> NaCl </td><td> 시그마 - 올드 리치 </td><td> 71,378 </td><td></td></tr><tr><td> KCl </td><td> 시그마 - 올드 리치 </td><td> 60,128 </td><td></td></tr><tr><td> Pyruvic 산 </td><td> 시그마 - 올드 리치 </td><td> P4562 </td><td></td></tr><tr><td> Hepes </td><td> 시그마 - 올드 리치 </td><td> 54,457 </td><td></td></tr><tr><td> MgCl 2 </td><td> 시그마 - 올드 리치 </td><td> M1020 </td><td></td></tr><tr><td> 아니 2 PO 4 일염기의 </td><td> 시그마 - 올드 리치 </td><td> P9791 </td><td></td></tr><tr><td> 우선당 </td><td> 시그마 - 올드 리치 </td><td> G6721 </td><td></td></tr><tr><td> NaOH </td><td> 시그마 - 올드 리치 </td><td> 72,068 </td><td></td></tr><tr><td> EGTA </td><td> 시그마 - 올드 리치 </td><td> O3777 </td><td></td></tr><tr><td> CaCl 2 </td><td> 시그마 - 올드 리치 </td><td> 21,115 </td><td></td></tr><tr><td> 단백 분해 효소, 유형 XIV </td><td> 시그마 - 올드 리치 </td><td> P5147 </td><td></td></tr><tr><td> Collagenase, 전을 입력합니다 </td><td> 워싱턴 </td><td> C9891 </td><td></td></tr><tr><td> 1X의 DNA 서스펜션 버퍼 (10 MM 트리스, 산도 8.0, 0.1 MM EDTA (에틸렌 다이아 민 테트라 초산)) </td><td> TEKnova, </td><td> PN T0221 </td><td></td></tr><tr><td> BSA </td><td> 시그마 - 올드 리치 </td><td> B8667 </td><td></td></tr><tr><td> Pentobarbital 나트륨 </td><td> 헨리 Schein의 투어뇌려. 공급 * </td><td> 주문에 대한 공급 업체에 문의하십시오 </td><td> *는 수의사 또는 동물 전문 연구소를 통해 procured되어야합니다 </td></tr><tr><td> IT를 ExoSAP </td><td> Affymetrix / USB </td><td> 78,201 </td><td></td></tr><tr><td> 2X Rxn 믹스를 CellsDirect </td><td> Invitrogen </td><td> 11737-030 </td><td></td></tr><tr><td> 윗첨자 III RT 플래티넘 DNA 형성 촉매 믹스 </td><td> Invitrogen </td><td> 10928-042 </td><td></td></tr><tr><td> RT - STA의 뇌관 믹스 </td><td> IDT </td><td> 사용자 정의 </td><td></td></tr><tr><td> Nuclease 무료 물 </td><td> 시그마 - 올드 리치 </td><td> W4502 </td><td></td></tr><tr><td> WTA2 </td><td> 시그마 - 올드 리치 </td><td> WTA2 - 50rxn </td><td></td></tr><tr><td> Qiaquick PCR 정화 키트 </td><td> Qiagen </td><td> 28,104 </td><td></td></tr><tr><td> Qiacube </td><td> Qiagen </td><td> 9,001,292 </td><td></td></tr><tr><td> NanoDrop 분광 광도계 </td><td>NanoDrop </td><td> 2000c </td><td></td></tr><tr><td> BioAnalyzer DNA 7500 키트 </td><td> 애질런트 </td><td> 7500 키트 </td><td></td></tr><tr><td> 한 컬러 라벨링 키트 </td><td> 호슈 - NimbleGen </td><td> 5223555001 </td><td></td></tr><tr><td> 뮤스 Musculus 12x135k 어레이 </td><td> 호슈 - NimbleGen </td><td> 5543797001 </td><td></td></tr><tr><td> GenePix 4200A 스캐너 </td><td> 분자 장치 </td><td> 4200A </td><td></td></tr><tr><td> TransPlex 완전한 전체 Transcriptome 증폭 키트 (WTA2 키트) </td><td> 시그마 - 올드 리치 </td><td> WTA2 - 50RXN </td><td></td></tr></tbody></table>

single cell transcriptional profiling of adult mouse cardiomyocytes

수많은 연구 심장 조직의 homogenates에서 유전자 발현 변화를 조사하고 있지만, 이것은 조직 내의 세포 사이의 고유의 확률적 변화를 공부하고 방지할 수 있습니다. 마우스 마음 collagenase 재관류를 통해 순수한 cardiomyocyte 인구의 격리는 전체 transcriptome 유전자 발현에 대한 단세포 microarrays의 생성, 또는 nanofluidic 배열을 사용하여 특정 대상의 qPCR을 용이하게합니다. 우리는 여기서 마음에서 격리 cardiomyocytes의 단일 세포 유전자 발현 프로파일을 조사하는 절차를 설명합니다. 이 패러다임은 유전자 발현의 평가 (그림 1) 종래의 전체 조직 워크플로를 사용 가능한 경우는 아니지만 (조직 내의 세포 사이의 예제 분산을위한) 의미에 의존하지 않습니다 흥미 통계 평가 수 있습니다. 우리는 전에 microarrays의 사용을 용이하게 이중 좌초 cDNA의 단일 세포 transcriptome 항복 마이크로 그램의 강력한 증폭을 달성ndividual 세포. 절차에서는 사용하고 자신의 디자인을 사용자 정의할 수있는 기능들의 편리를 위해 선택되었다 NimbleGen 배열의 사용을 설명합니다. 또는 반대 transcriptase - 구체적인 목표 증폭 (RT - STA) 반응, nanofluidic PCR에 의한 대상의 수백 qPCR을 허용합니다. 어느 이러한 방식을 사용하여, 그것은 세포 사이의 표현의 다양성뿐만 아니라, 조직 내에서 드문 세포 유형의 표현 프로필을 검토을 검토하는 것이 가능합니다. 전반적으로, 단일 세포 유전자 표현 방식은 잠재적으로 전체 조직에서 생성된 전형적인 homogenates의 세포 수백만의 표현을 조사했을 때 일반적으로 밖으로 평균 아르 특이한 표현 프로파일을 식별할 수있는 데이터의 생성을 허용합니다.

Watch this Scientific Journal Video about Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes at JoVE.com

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

단세포 표현 프로 파일링 개별 세포의 자세한 유전자 발현 분석을 수 있습니다. 우리는 cardiomyocytes의 격리를 위해 방법을 설명하고, 구체적인 목표의 전체 transcriptome의 microarray 또는 qPCR 중 하나에 대한 결과 lysates를 준비.

성인 마우스 Cardiomyocytes의 단세포 Transcriptional 프로파일

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

single-cell-transcriptional-profiling-of-adult-mouse-cardiomyocytes

Research

JoVE Journal

Biology

17.2K 조회수.  Buck Institute for Research on Aging. 단세포 표현 프로 파일링 개별 세포의 자세한 유전자 발현 분석을 수 있습니다. 우리는 cardiomyocytes의 격리를 위해 방법을 설명하고, 구체적인 목표의 전체 transcriptome의 microarray 또는 qPCR 중 하나에 대한 결과 lysates를 준비.

비디오: Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

분리 및 문화 신생아 마우스 심근

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

연구

생물학

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

마우스 심근의 분리 및 생리 학적 분석

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

분리 및 문화 성인 마우스 세포 신호 전달 용의 Cardiomyocytes과의<em&gt; 시험관</em&gt; 심장 비대

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

개별 세포 유형의 전사 프로파일 링을위한 도구로 레이저를 이용한 이외에 Microdissection (LAM)

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

격리, 문화 및 성인 마우스 심근 세포의 형질 도입

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

트랜스-내부 세포 배아 줄기 세포의 대량 주입 리드 더 높은 Chimerism 요금

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying cell composition, discrimination of functional states, and molecular pathways underlying observed tissue functions and animal behaviors. However, the isolation of intact, healthy single cells from adult mammalian tissues for subsequent downstream single cell molecular analysis can be challenging. This protocol describes the general processes and quality control checks necessary to obtain high-quality adult single cell preparations from the nervous system or skin that enabled subsequent unbiased single cell RNA sequencing and analysis. Guidelines for downstream bioinformatic analysis are also provided.

This protocol describes the general processes and quality control checks necessary for preparing healthy adult mammalian single cells for droplet-based, high throughput single cell RNA-Seq preparations. Sequencing parameters, read alignment, and downstream single-cell bioinformatic analysis are also provided.

드롭릿 바코드 기반 단일 셀 Transcriptomics 성인 포유류 직물의

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

마우스 간에서 세포 유형 별 유전자 발현 프로파일링

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

성인 마우스및 쥐 심근세포의 격리, 트랜스페션 및 장기 문화

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &amp;#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

단일 분자 국소화 현미경 검사를 사용하여 인간 iPSC 파생 심근세포에서 α-액티닌 네트워크 분석

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...