우리는 읽는이 원고의 편집을 위해 닥터 노아 Weisleder 감사드립니다. 이 작품은 메가바이트에 0535555N ZP, XZ로 미국 심장 협회 SDG2630086, 그리고 건강의 국립 연구소 RC2AR058962-01메가바이트까지 연구 보조금 UMDNJ 재단 62-09에 의해 지원되었다.

<ol>
	<li>Parekh, A. B., Putney, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Store-operated+calcium+channels.">Store-operated calcium channels.</a> Physiol. Rev. 85, 757-810 (2005).</li><li>Ma, J., Pan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+activation+of+store-operated+calcium+channel.">Retrograde activation of store-operated calcium channel.</a> Cell Calcium. 33, 375-384 (2003).</li><li>Feske, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ORAI1+and+STIM1+deficiency+in+human+and+mice:+roles+of+store-operated+Ca2++entry+in+the+immune+system+and+beyond.">ORAI1 and STIM1 deficiency in human and mice: roles of store-operated Ca2+ entry in the immune system and beyond.</a> Immunol. Rev. 231, 189-209 (2009).</li><li>Parekh, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Store-operated+CRAC+channels:+function+in+health+and+disease.">Store-operated CRAC channels: function in health and disease.</a> Nat. Rev. Drug Discov. 9, 399-410 (2010).</li><li>Pan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysfunction+of+store-operated+calcium+channel+in+muscle+cells+lacking+mg29.">Dysfunction of store-operated calcium channel in muscle cells lacking mg29.</a> Nat. Cell Biol. 4, 379-383 (2002).</li><li>Shin, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+retrograde+signal+from+calsequestrin+for+the+regulation+of+store-operated+Ca2++entry+in+skeletal+muscle.">A retrograde signal from calsequestrin for the regulation of store-operated Ca2+ entry in skeletal muscle.</a> J. Biol. Chem. 278, 3286-3292 (2003).</li><li>Ma, J., Pan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Junctional+membrane+structure+and+store+operated+calcium+entry+in+muscle+cells.">Junctional membrane structure and store operated calcium entry in muscle cells.</a> Front Biosci. 8, d242-d255 (2003).</li><li>Pan, Z., Damron, D., Nieminen, A. L., Bhat, M. B., Ma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+intracellular+Ca2++by+caffeine+and+ryanodine+induces+apoptosis+of+chinese+hamster+ovary+cells+transfected+with+ryanodine+receptor.">Depletion of intracellular Ca2+ by caffeine and ryanodine induces apoptosis of chinese hamster ovary cells transfected with ryanodine receptor.</a> J. Biol. Chem. 275, 19978-19984 (2000).</li><li>Hirata, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncoupling+store-operated+Ca2++entry+and+altered+Ca2++release+from+sarcoplasmic+reticulum+through+silencing+of+junctophilin+genes.">Uncoupling store-operated Ca2+ entry and altered Ca2+ release from sarcoplasmic reticulum through silencing of junctophilin genes.</a> Biophys. J. 90, 4418-4427 (2006).</li><li>Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+resistance+to+fatigue+and+altered+calcium+handling+properties+of+sarcalumenin+knockout+mice.">Enhanced resistance to fatigue and altered calcium handling properties of sarcalumenin knockout mice.</a> Physiol. Genomics. 23, 72-78 (2005).</li><li>Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Azumolene+inhibits+a+component+of+store-operated+calcium+entry+coupled+to+the+skeletal+muscle+ryanodine+receptor.">Azumolene inhibits a component of store-operated calcium entry coupled to the skeletal muscle ryanodine receptor.</a> J. Biol. Chem. 281, 33477-33486 (2006).</li><li>Launikonis, B. S., Barnes, M., Stephenson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+coupling+between+skeletal+muscle+store-operated+Ca2++entry+and+the+inositol+trisphosphate+receptor.">Identification of the coupling between skeletal muscle store-operated Ca2+ entry and the inositol trisphosphate receptor.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 2941-2944 (2003).</li><li>Shuttleworth, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+relationships+between+Ca2++store+mobilization+and+Ca2++entry+in+an+exocrine+cell.">Temporal relationships between Ca2+ store mobilization and Ca2+ entry in an exocrine cell.</a> Cell. Calcium. 15, 457-466 (1994).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

칼슘 2 + 바인딩 및 CA 2 + 무료 Fura-2의 여기 파장은 각각 340 nm의 380 nm의 있지만, CA 2 Fura-2의 최고 비율 동적 범위는 + 농도 측정은 특히 다른 파장에서 발생할 수 현미경 시스템. 파장에서의 이러한 변화는 일반적으로 다양한 광학 부품의 추가와 광학 경로의 변화로 인해 수 있습니다. 본 연구에서는 Fura-2의 여기 파장은 Fura-2 형광 분광 분석을 수행하여 350 nm의 및 390 nm의로 결정됩니다. 를 포함한 여러 소스의 균형에서 cytosol 결과에서 세포 내 칼슘 2 + 수준의 세포내 칼슘 2 + 릴리스, 세포외 칼슘 2 + 항목뿐만 아니라 응급실과 플라즈마 막에서 칼슘 2 + 배제 메커니즘. 세포내 칼슘 2 + 릴리스 및 CA 2 + extr에서 단방향 SOC-매개 칼슘 2 + 유입을 분리하려면usion는 MN 2 + 담금질 분석을 사용할 수 있습니다. MN 2 +는 SOCE 통해 세포에 침투 수 있도록 알려져 있지만 표면 막 압출 또는 칼슘이에 의해 응급실로 이해 + 펌프를 안타입니다. 따라서 형광 담금질은 SOCE의 활성화 정도를 예상 세포로 단방향 MN 2 + 유량의 측정을 나타냅니다. MN 2 + 담금질 분석 같은 파장에서 isosbestic 시점 공연 Fura-2 형광 강도는 칼슘 2 + 농도에 독립적입니다.합니다 각 시스템에 대한 isosbestic 포인트는 스펙트럼 스캔에 의해 결정되어야한다. 또한, MN 2 + 담금질은 최종 값이 칼슘 2 + 농도 13 독립이라는 같은 방법으로 두 개의 파장에서 빛을 조정 형광에 의해 기록될 수 있습니다. 골격 근육의 SOCE의 활동 공간과 시간적 정보를 얻으려면 우리는 동시 measureme 수 있도록 이중 염색 방법을, 고용SOCE 활동 및 SR 칼슘이 피부 성인 포유류의 편집 판단리스트 근육 섬유에 + 컨텐츠 NT. SR 칼슘 2의 변화 사이의 상관 관계는 + 내용 및 SOCE 활동은 SR 칼슘 2의 고갈 + 스토리지로 SOCE 활성화의 한계와 감도를 나타내는 꾸몄다 수 있습니다. TT-로드 솔루션은 또한 칼슘 2 T-tubules을로드하는 데 최적화되어 있습니다 +와 T-tubules 및 SR-로딩 솔루션의 마중물을 위해 최대한의 SR 칼슘 2 + 로딩을 촉진하고 추가로 T-tubules를 로드할 수 있도록 설계되었습니다 ~ 500 μm의 칼슘 2 +. FCCP는 TT / SR-로딩 솔루션과 mitochondria의 효과를 제거하는 SR-파괴 솔루션에 포함되어 있습니다.

특정 시약 및 장비의 테이블 : <table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> RPMI 1640 </td><td> Mediatech </td><td> 10-040 - 이력서 </td><td></td></tr><tr><td> 햄의 F-12 </td><td> Mediatech </td><td> 10-080 - 이력서 </td><td></td></tr><tr><td> DMEM </td><td> Mediatech </td><td> 10-013 - 이력서 </td><td></td></tr><tr><td> 유리 바닥 요리 </td><td> MatTek </td><td> P35G-1.5-14-C </td><td></td></tr><tr><td> Fura - 오전 2시 </td><td> Invitrogen (분자 프로브) </td><td> F1221 </td><td> -20 ° C, DMSO에 녹아있는 빛으로부터 보호 도장 꽉,, </td></tr><tr><td> Fluo-5N 오전 </td><td> Invitrogen (분자 프로브) </td><td> F14204 </td><td> -20 ° C, 도장 꽉 전로부터 보호항공 </td></tr><tr><td> 로드-5N </td><td> Invitrogen (분자 프로브) </td><td> R14207 </td><td> -20 ° C, 도장 꽉은 빛으로부터 보호 </td></tr><tr><td> 석영 Cuvette </td><td> Starna 셀 </td><td> 3-Q-10 </td><td></td></tr><tr><td> thapsigargin </td><td> Tocris </td><td> 1138 </td><td></td></tr><tr><td> 2-Aminoethoxydiphenylborane (2-지명수배) </td><td> Tocris </td><td> 1224 </td><td></td></tr><tr><td> N-벤질-P-톨루엔 sulphonamide (BTS) </td><td> 시그마 - 올드 리치 </td><td> 435,600 </td><td></td></tr><tr><td> Collagenase 유형 I </td><td> 시그마 </td><td> C0130 </td><td></td></tr></tbody></table> 장비 : <ol><li> spectrofluorometer (광자 기술 인터내셔널, 뉴저지) : 크세논 아크 램프, 컴퓨터 제어 고속 랜덤 액세스 단색, cuvette 기반 방출 단색, 니콘 TE-200 거꾸로 형광 microscopE, S 형석 40x/1.30 오일 목적, PMT 탐지기. </li><li> 아르곤 레이저 453/477/488/514, HeNe 레이저 543nm, 니콘 TE2000 거꾸로 현미경, 60X 목표 (NA 1.4, 석유) : 레이저 공촛점 현미경 (BioRad Radiance210​​0)를 스캔. </li><li> 배율 파워&gt; 100 (자이스 혈구 Stemi SV11 APO)와 stereomicroscope. </li></ol>

fluorescence-based measurement of store-operated calcium entry in live cells: from cultured cancer cell to skeletal muscle fiber

쇼핑몰 이전 capacitative 칼슘 2 + 항목을 되나, CA 2 + 항목 (SOCE)를 운영, + 고갈 endoplasmic reticulum (ER) 또는 sarcoplasmic reticulum (SR) 칼슘 2 + 저장소를 보충하기 위해 세포에 세포 밖 칼슘 2의 유입을위한 단단히 규제 메커니즘입니다 1,2. 칼슘 2 + 유비 쿼터스 초 메신저이기 때문에, 그 SOCE가 증식, apoptosis, 유전자 전사 및 운동성 포함한 세포 프로세스 다양한 중요한 역할을 담당 볼 놀라운 일이 아닙니다. 상피 세포와 골격 근육을 포함한 거의 모든 세포 유형에서 자사의 광범위한 발생으로 인해,이 통로가 큰 관심 3,4을 받았습니다. 그러나, 다른 세포 유형 및 생리적 기능에 SOCE 특징 이질 아직도 5-7 해제되지 않습니다. SOCE의 기능적 채널 속성이 생에 대한 지식의 큰 몸 반면, 패치 - 클램프 연구에 의해 밝혀하실 수 있습니다의 경로가 있기 때문에 높은 처리량 검사를위한 편리 성과 타당성의 형광 기반의 세포내 칼슘 2 + 측정하여 얻고있다. 이 보고서의 목적은 단일층 세포, 정지 세포와 근육 섬유 5,8-10의 SOCE의 활성을 측정하기 위하여 몇 가지 형광 기반의 방법을 요약하라는 것입니다. 가장 일반적으로 이러한 형광 방법 중 하나를 사용하는 직접 세포내 칼슘 2의 역학을 모니터 + F를 340nm와 ratiometric 칼슘 2 + 지표 Fura-2의 F 380nm (발광 파장에 대한 510 nm의)의 비율을 사용하고 있습니다. 세포내 칼슘 2 + 릴리스 및 CA 2에서 단방향 SOCE의 활동을 분리하려면 + 압출는 MN 2 + 담금질 분석이 자주 사용됩니다. MN 2 +는 그 표면 막 압출 공정이나 칼슘이에 의해 응급실로 이해를 안타 중에 SOCE 통해 세포에 침투 수 있도록 알려져있다 + 매우 높은 친화도 재치로 인해 펌프는H Fura - 2. 따라서 + 세포에 세포 MN 2의 항목에 의해 유도된 Fura-2 형광 담금질은 SOCE 9 활동의 측정을 나타냅니다. Ratiometric 측정 및 MN이 담금질의 assays는 세포 인구 모드에서 단일 세포를 시각화하는 현미경 기반 시스템의 cuvette 기반 spectrofluorometer 수행할 수 있습니다. 단일 세포 측정의 장점은 유전자 조작에 노출 개별 세포가 유전자 변형 또는 변이되는 세포의 연구를 수 있도록 GFP 혹은 RFP 기자를 사용하여 선택할 수있다는 것입니다. 구조적으로 전문 골격근의 SOCE의 spatiotemporal 특성은 동시에이 낮은 친화력 칼슘 2의 형광을 모니터하여 피부 근육 섬유에서 얻을 수 SR과 이러한 Fluo-5N과 같은 근육 섬유의 특정 구획, 타겟으로 + 지표로드 - 가로 tubules 9,11,12에서 5N.

Watch this Scientific Journal Video about Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber at JoVE.com

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

매장 운영하는 CA의 범위<sup&gt; 2 +</sup&gt; 항목 (SOCE)는 형광 칼슘을 사용하여 모니터링할 수 있습니다<sup&gt; 2 +</sup&gt; 지표. MN<sup&gt; 2 +</sup교양 세포와 골격근 섬유의 이러한 지표의 assays의 SOCE의&gt; 담금질. 기계적으로 피부를 근육 섬유의 공촛점 이미징에 의한 SOCE의 공간과 시간적 해상도를 허용하는 방법도 설명되어 있습니다.

라이브 세포의 저장소로 작동하는 칼슘 항목의 형광 기반의 측정 : 교양 암 세포에서 골격근 섬유에

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

fluorescence-based-measurement-store-operated-calcium-entry-live

Research

JoVE Journal

Biology

21.0K 조회수.  Robert Wood Johnson Medical School. 매장 운영하는 CA의 범위 2 + 항목 (SOCE)는 형광 칼슘을 사용하여 모니터링할 수 있습니다 2 + 지표. MN 2 + 담금질. 기계적으로 피부를 근육 섬유의 공촛점 이미징에 의한 SOCE의 공간과 시간적 해상도를 허용하는 방법도 설명되어 있습니다.

비디오: Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Mitochondrial Isolation from Skeletal Muscle

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

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

골격근에서 Mitochondrial 절연

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

연구

생물학

Purification of Progenitors from Skeletal Muscle

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

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

골격근에서 Progenitors의 정화

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

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

성인 골격근의 개인 Myofibers 및 위성 세포의 분리와 문화

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

칼슘의 평가는 그대로 골격 근육 섬유에서 불꽃

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

인간의 골격 근육에서 혈관 유래 다 능성 전구체의 분리

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

배양 된 평활근 세포의 근질 그물 자리 내 칼슘 변동 FRET 측정을 기반으로 공 촛점 이미지

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

형광 현미경으로 살아있는 세포의 잠재적 인 미토콘드리아의 칼슘과 미토콘드리아 막의 동시 측정

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins

Small cysteine-rich defensins are one of the largest groups of host defense peptides present in all plants. Many plant defensins exhibit potent in vitro antifungal activity against a broad-spectrum of fungal pathogens and therefore have the potential to be used as antifungal agents in transgenic crops. In order to harness the full potential of plant defensins for diseases control, it is crucial to elucidate their mechanisms of action (MOA). With the advent of advanced microscopy techniques, live-cell imaging has become a powerful tool for understanding the dynamics of the antifungal MOA of plant defensins. Here, a confocal microscopy based live-cell imaging method is described using two fluorescently labeled plant defensins (MtDef4 and MtDef5) in combination with vital fluorescent dyes. This technique enables real-time visualization and analysis of the dynamic events of MtDef4 and MtDef5 internalization into fungal cells. Importantly, this assay generates a wealth of information including internalization kinetics, mode of entry and subcellular localization of these peptides. Along with other cell biological tools, these methods have provided critical insights into the dynamics and complexity of the MOA of these peptides. These tools can also be used to compare the MOA of these peptides against different fungi.

Plant defensins play an important role in plant defense against pathogens. For effective use of these antifungal peptides as antifungal agents, understanding their modes of action (MOA) is critical. Here, a live-cell imaging method is described to study critical aspects of the MOA of these peptides.

조사 항목의 모드와 항진균 공장 Defensins의 Subcellular 지 방화 하는 곰 팡이 세포의 라이브 셀 이미징

Small cysteine-rich defensins are one of the largest groups of host defense peptides present in all plants. Many plant defensins exhibit potent in vitro ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Ca2+ 과도 현상 및 L형 칼슘 전류의 동시 측정을 위한 고품질 쥐 심방 및 심실 근세포의 분리

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment failure is mainly due to the invasiveness of GBM cells, which speaks for the need for a better understanding of GBM motile properties. To investigate the molecular mechanism supporting GBM invasion, new physiological models enabling in-depth characterization of protein dynamics during invasion are required. These observations would pave the way to the discovery of novel targets to block tumor infiltration and improve patient outcomes. This paper reports how an orthotopic xenograft of GBM cells in the zebrafish brain permits subcellular intravital live imaging. Focusing on microtubules (MTs), we describe a procedure for MT labeling in GBM cells, microinjecting GBM cells in the transparent brain of 3 days post fertilization (dpf) zebrafish larvae, intravital imaging of MTs in the disseminating xenografts, altering MT dynamics to assess their role during GBM invasion, and analyzing the acquired data.

We report a technique permitting live imaging of microtubule dynamics in glioblastoma (GBM) cells invading a vertebrate brain tissue. Coupling the orthotopic injection of fluorescently tagged GBM cells into a transparent zebrafish brain with high-resolution intravital imaging allows the measurement of cytoskeleton dynamics during in situ cancer invasion.

제브라피쉬의 뇌를 침범하는 교모세포종 세포의 미세소관 역학의 라이브 이미징

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment ...