私たちは、読書と、この原稿を編集するために博士ノアWeislederに感謝します。この作品は、MBに0535555N ZP、XZに米国心臓協会SDG2630086、および国立衛生研究所RC2AR058962-01 MBまでに研究費補助金UMDNJ財団62から09でサポートされていました。

<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>

利害の衝突が宣言されません。

Ca 2 +結合およびCa 2 +フリーフラ-2の励起波長は、それぞれ340nmおよび380nmであるが、Caの2のフラ-2の最高の比ダイナミックレンジ+濃度測定は、特に、他の波長で発生する可能性があります顕微鏡システム。波長のようなシフトは、通常、様々な光学部品を添加した光路の変化によるものである。本研究では、フラ-2の励起波長は、フラ-2蛍光のスペクトル解析を行うことにより、350 nmと390 nmで測定した。 を含むいくつかのソースのバランスから、細胞質ゾルの結果細胞内Ca 2 +レベル、細胞内Ca 2 +放出は、細胞外Ca 2 +のエントリと同様に、小胞体と細胞膜でのCa 2 +排除機構。細胞内Ca 2 +放出とCa 2 + EXTRから一方向のSOC-介在Ca 2 +流入を単離するために、usionは、Mn 2 +が消光アッセイを使用することができます。のMn 2 +は SOCEを介して細胞に浸透することができることが知られているが、表面膜の押し出しまたはCa 2によってERの取り込み+ポンプへの浸透です。したがって、蛍光消光は、単方向のMn 2 + SOCEの活性化の度合いを推定する細胞内へのフラックスの測定を表しています。のMn 2 +クエンチアッセイは、このような波長で、等吸収点で行わフラ-2蛍光強度がCa 2 +濃度とは無関係です。されてい各システムの等吸収点は、スペクトラム·スキャンによって決定されるべきである。また、Mn 2 +の消光は、最終的な値は、Ca 2 +濃度の13の独立しているような方法で、任意の2つの波長で調整された蛍光によって記録することができます。 骨格筋におけるSOCEの活動の空間的および時間的情報を得るために、我々は同時measuremeを可能にするデュアル色素法を採用SOCE活動のNTおよびSRのCa 2 +肌の大人の哺乳類のEDL筋線維内のコンテンツ。 SRのCa 2の変化の間に相関が+コンテンツとSOCE活動は、SRのCa 2 +貯蔵の枯渇にSOCE活性化のしきい値と感度を示すためにプロットすることができます。 TT-ローディング溶液は、さらにカルシウム2とT-細管をロードするために最適化されている+とT-細管とSR-ローディング溶液のプライミングのために最大限のSRのCa 2 +ローディングを促進し、さらにT-細管をロードするように設計されています〜500μMのCa 2 +。 FCCPは、TT / SR-ローディング溶液とミトコンドリアからの影響を排除するためにSR-オゾンソリューションに含まれています。

特定の試薬や機器の表： <table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> RPMI 1640 </td><td>メディアテック</td><td> 10から040-CV </td><td></td></tr><tr><td>ハムのF-12 </td><td>メディアテック</td><td> 10から080-CV </td><td></td></tr><tr><td> DMEM </td><td>メディアテック</td><td> 10から013-CV </td><td></td></tr><tr><td>ガラスボトムディッシュ</td><td>マテック</td><td> P35G-1.5-14-C </td><td></td></tr><tr><td> AMフラ-2 </td><td> Invitrogen社（Molecular Probes）を</td><td> F1221 </td><td> -20°C、DMSOに溶解し、光から保護シールタイト、 </td></tr><tr><td>蛍光-5N AM </td><td> Invitrogen社（Molecular Probes）を</td><td> F14204 </td><td> -20°C、密封、Lから保護IGHT </td></tr><tr><td> rhodo-の異形5N </td><td> Invitrogen社（Molecular Probes）を</td><td> R14207 </td><td> -20°C、シールタイト、光から保護</td></tr><tr><td>石英キュベット</td><td> Starna細胞</td><td> 3-Q-10 </td><td></td></tr><tr><td>タプシガルギン</td><td> Tocris </td><td> 1138 </td><td></td></tr><tr><td> 2 Aminoethoxydiphenylborane（2-APB） </td><td> Tocris </td><td> 1224 </td><td></td></tr><tr><td> N-ベンジル-p-トルエンスルホンアミド（BTS） </td><td> Sigma-Aldrich社</td><td> 435600 </td><td></td></tr><tr><td>コラゲナーゼタイプI </td><td>シグマ</td><td> C0130 </td><td></td></tr></tbody></table>設備： <ol><li>分光蛍光光度計（フォトンテクノロジーインターナショナル、NJ）：キセノンアークランプ、コンピュータ制御の高速ランダム·アクセス·モノクロ、キュベットベースの発光分光、ニコンTE-200倒立蛍光microscopE、S弗素40x/1.30油浸対物、PMT検出器。 </li><li>アルゴンレーザー453/477/488/514、HeNeレーザー543nm、ニコン倒立顕微鏡TE2000、60X対物レンズ（NA 1.4、油）：レーザー共焦点顕微鏡（BioRad社Radiance210​​0）をスキャンしています。 </li><li>倍率の実体顕微鏡&gt; 100（ツァイスSTEMI SV11 APO）。 </li></ol>

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

店は以前の容量性Ca 2 +のエントリと呼ばれる、Ca 2 +のエントリ（SOCE）を運営し、+枯渇小胞体（ER）や筋小胞体（SR）のCa 2 +ストアを補充する細胞に細胞外Ca 2 +の流入のために厳密に制御メカニズムです。 1,2。のCa 2 +ユビキタスセカンドメッセンジャーであるので、SOCEが増殖、アポトーシス、遺伝子の転写と運動性を含む細胞プロセスの様々な重要な役割を果たしているのを見て驚くことではありません。上皮細胞と骨格筋を含むほぼすべての細胞型で広い発生のため、この経路は、大きな関心3,4を受けています 。ただし、異なる細胞型と生理機能のSOCE特性の不均一性は、まだ5-7クリアされていません。 SOCEの機能チャネルのプロパティは、THIに関する知識の大きな体に対し、パッチクランプ法の研究によって明らかにすることができるの経路があるため、ハイスループットスクリーニングのため、その利便性と実現可能性の蛍光ベースの細胞内Ca 2 +測定によって得られている。このレポートの目的は、単層細胞、浮遊細胞や筋線維5,8-10でSOCEの活性化を測定するためにいくつかの蛍光ベースの方法を要約することである。最も一般的にこれらの蛍光法の使用は、直接細胞内Ca 2 +の動態を監視するために+ F 340nm、レシオメトリックのCa 2 +インジケーターフラ-2のF 波長380nm（発光波長510 nm）の比を使用しています。細胞内Ca 2から一方向SOCEの活性を分離するには+の放出とCa 2 +放出は、Mn 2 +クエンチアッセイが頻繁に使用されます。それは非常に高い親和性ウィットのために+ポンプ表面膜の押出プロセスまたはCa 2によってERの取り込みに不浸透性のある間のMn 2 +は SOCEを介して細胞に浸透することができることが知られているHフラ-2。その結果、細胞内に細胞外のMn 2 +のエントリによって誘発されるフラ-2蛍光の消光がSOCE 9の活性の測定を表しています。レシオメトリック測定とMn 2消光アッセイは、単一細胞を可視化する細胞集団モードまたは顕微鏡ベースのシステムでキュベットベースの蛍光光度計で実行することができます。単一細胞の測定の利点は、遺伝子操作を受け、個々の細胞は、遺伝子組み換えや変異細胞の研究をできるように、GFPまたはRFPレポーターを使用して選択することができることである。構造的に特殊な骨格筋でSOCEの時空間特性は、同時に二つの低親和性Ca 2 +の蛍光を監視することによって、肌の筋線維で達成することができるSRと、このようなにみたFluo-5Nとして筋線維の特定のコンパートメントを対象とした+の指標rhodo-の異形横細管9,11,12で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

ストア作動性Caの範囲<sup&gt; 2 +</sup&gt;エントリ（SOCE）は、蛍光Caを使用して監視することができます<sup&gt; 2 +</sup&gt;指標。 Mnは<sup&gt; 2 +</sup培養細胞や骨格筋線維のような指標アッセイSOCEの&gt;消光。機械的に皮筋線維の共焦点イメージングによるSOCEの空間分解能と時間分解能を可能にする技術も記載されています。

生細胞におけるストア作動性カルシウムエントリの蛍光ベースの測定：培養癌細胞から骨格筋線維に

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 ビュー.  Robert Wood Johnson Medical School. ストア作動性Caの範囲 2 +エントリ（SOCE）は、蛍光Caを使用して監視することができます 2 +指標。 Mnは 2 +消光。機械的に皮筋線維の共焦点イメージングによるSOCEの空間分解能と時間分解能を可能にする技術も記載されています。

ビデオ: 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.

骨格筋からのミトコンドリア単離

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

生物学

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.

骨格筋からの前駆体の精製

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.

成人骨格筋からの個々の筋線維とその衛星細胞の単離および培養

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.

無傷骨格筋線維内のカルシウムスパークスの評価

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.

ヒト骨格筋からの血管由来の多能前駆体の単離

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.

FRETベースの共焦点イメージングを使用した培養平滑筋細胞の小胞体内カルシウム変動の測定

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.

蛍光顕微鏡による生細胞内のミトコンドリアのカルシウムとミトコンドリア膜電位の同時測定

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.

エントリのモードと抗真菌植物のディフェンシンの細胞内局在を検討する真菌細胞の生細胞イメージング

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+過渡現象とL型カルシウム電流の同時測定のための高品質マウス心房および心室筋細胞の分離

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.

ゼブラフィッシュ脳に侵入する膠芽腫細胞における微小管動態のライブイメージング

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