Wir danken Dr. Noah Weisleder zum Lesen und Editieren von diesem Manuskript. Diese Arbeit wurde durch Forschungsstipendien UMDNJ Stiftung 62-09 bis ZP, American Heart Association SDG2630086 zu XZ, 0535555N zu MB, und National Institutes of Health RC2AR058962-01 bis MB unterstützt.

<ol>
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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>

Keine Interessenskonflikte erklärt.

Obwohl die Anregungswellenlängen für Ca 2 +-Bindung und Ca 2 +-freien Fura-2 340 nm und 380 nm sind jeweils das beste Verhältnis Dynamikbereich von Fura-2 für Ca 2 +-Konzentration Messung kann bei anderen Wellenlängen bei einer bestimmten Mikroskop-System. Solche Verschiebungen in der Wellenlänge sind in der Regel aufgrund von Änderungen in dem optischen Pfad mit der Zugabe von verschiedenen optischen Komponenten. In dieser Studie wurden die Anregungswellenlängen für Fura-2 als 350 nm und 390 nm zur Spektralanalyse von Fura-2 Fluoreszenz bestimmt. Die intrazellulären Ca 2 +-Ebene im Zytosol resultiert aus einem Gleichgewicht aus mehreren Quellen, einschließlich intrazellulärer Ca 2 +-Freisetzung, extrazellulären Ca 2 +-Eintrag, sowie Ca 2 +-Ausschluss-Mechanismus an der ER und Plasmamembran. Um die unidirektionale SOC-vermittelte Ca 2 +-Einstrom aus intrazellulären Ca 2 +-Freisetzung und Ca 2 + extr isolierenusion, kann die Mn 2 + Abschrecken des Tests verwendet werden. Mn 2 + ist bekannt, können in die Zellen über SOCE Permeat aber undurchlässig für Oberflächenmembran Extrusion oder ER Aufnahme von Ca 2 +-Pumpen. Daher stellt Fluoreszenzlöschung eine Messung der unidirektionalen Mn 2 +-Fluss in die Zellen, die den Grad der Aktivierung des SOCE schätzt. Mn 2 + Abschrecken Test wird bei der isosbestischen Punkt durchgeführt, an solchen Wellenlänge Fura-2-Fluoreszenzintensität ist unabhängig von Ca 2 +-Konzentration. Die isosbestischen Punkt für jedes System sollte durch Scannen Spektrum bestimmt werden. Alternativ kann Mn 2 + Abschrecken durch angepasste Fluoreszenz bei zwei beliebigen Wellenlängen so dass der endgültige Wert unabhängig von Ca 2 +-Konzentration 13 aufgezeichnet werden. Um die räumlichen und zeitlichen Informationen der Tätigkeit der SOCE in der Skelettmuskulatur zu gewinnen, verwendeten wir eine Dual-Farbstoff-Methode, die gleichzeitige MeasureMe erlaubtnt von SOCE Aktivität und SR Ca 2 +-Gehalt in enthäuteten erwachsener Säugetiere EDL Muskelfasern. Die Korrelation zwischen den Veränderungen im SR Ca 2 +-Gehalt und SOCE Aktivität kann aufgetragen, um die Schwelle und Empfindlichkeit der SOCE Aktivierung zu einer Erschöpfung des SR Ca 2 +-Lagerung hinzuweisen. Der TT-loading Lösung ist optimiert, um zusätzlich laden die T-Tubuli mit Ca 2 + und für die Grundierung der T-Tubuli und der SR-loading-Lösung wurde entwickelt, um maximale SR Ca 2 +-Beladung zu fördern und zusätzlich laden die T-Tubuli mit ~ 500 uM Ca 2 +. FCCP befindet sich im TT / SR-loading-Lösung und der SR-abbauenden Lösung, um die Effekte aus den Mitochondrien eliminieren enthalten.

Tabelle der spezifischen Reagenzien und Geräte: <table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> RPMI 1640 </td><td> Mediatech </td><td> 10 bis 040-CV </td><td></td></tr><tr><td> Ham &#39;s F-12 </td><td> Mediatech </td><td> 10 bis 080-CV </td><td></td></tr><tr><td> DMEM </td><td> Mediatech </td><td> 10 bis 013-CV </td><td></td></tr><tr><td> Glass Bottom Dish </td><td> MatTek </td><td> P35G-1.5-14-C </td><td></td></tr><tr><td> Fura-2 AM </td><td> Invitrogen (Molecular Probes) </td><td> F1221 </td><td> -20 ° C, Dichtung eng, vor Licht geschützt, in DMSO gelöst </td></tr><tr><td> Fluo-5N Uhr </td><td> Invitrogen (Molecular Probes) </td><td> F14204 </td><td> -20 ° C, Dichtung dicht, von l geschütztight </td></tr><tr><td> Rhod-5N </td><td> Invitrogen (Molecular Probes) </td><td> R14207 </td><td> -20 ° C, Dichtung eng, vor Licht geschützt </td></tr><tr><td> Quarzküvette </td><td> Starna Cells </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-APB) </td><td> Tocris </td><td> 1224 </td><td></td></tr><tr><td> N-Benzyl-p-Toluolsulfonamid (BTS) </td><td> Sigma-Aldrich </td><td> 435600 </td><td></td></tr><tr><td> Kollagenase Typ I </td><td> Sigma </td><td> C0130 </td><td></td></tr></tbody></table> Ausstattung: <ol><li> Ein Spektrofluorometer (Photon Technology International, New Jersey): Xenon-Bogenlampe, computergesteuerten High-Speed-Random-Access-Monochromator, Küvetten-basierte Emissionsmonochromator, Nikon TE-200 invertierte Fluoreszenz-MikroskopE, S Fluor 40x/1.30 Öl-Objektiv, PMT-Detektoren. </li><li> Laser-Scanning-konfokalen Mikroskop (BioRad Radiance2100): 453/477/488/514 Argonlaser, HeNe-Laser 543nm, Nikon TE2000 inversen Mikroskop, 60x Objektiv (NA 1.4, Öl). </li><li> Ein Stereomikroskop mit einer Vergrößerungsleistung&gt; 100 (Zeiss Stemi SV11 Apo). </li></ol>

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

Shop betrieben Ca 2 +-Eintrag (SOCE), früher nannte kapazitiven Ca 2 +-Eintrag, ist ein stark regulierter Mechanismus zum Einstrom von extrazellulären Ca 2 + in Zellen mit abgereichertem Endoplasmatischen Retikulum (ER) oder sarkoplasmatischen Retikulum (SR) Ca 2 + wieder aufzufüllen 1,2. Da Ca 2 + ist ein allgegenwärtiges second messenger, ist es nicht überraschend zu sehen, dass SOCE spielt eine wichtige Rolle in einer Vielzahl von zellulären Prozessen wie Proliferation, Apoptose, Gen-Transkription und Motilität. Aufgrund ihrer weiten Verbreitung in nahezu allen Zelltypen, einschließlich Epithelzellen und Skelettmuskulatur, hat dieser Signalweg großes Interesse 3,4 erhielt. Allerdings sind die Heterogenität der SOCE Eigenschaften in verschiedenen Zelltypen und die physiologische Funktion noch nicht klar, 5-7. Die funktionellen Eigenschaften von Kanal SOCE kann durch Patch-Clamp-Studien enthüllt werden, während ein großer Teil der Kenntnisse über this-Mechanismus wurde durch Fluoreszenz-basierten intrazellulärer Ca 2 +-Messungen wegen seiner Bequemlichkeit und Machbarkeit für das Hochdurchsatz-Screening gewonnen werden. Das Ziel dieses Berichtes ist es, ein paar Fluoreszenz-basierten Methoden zusammenfassen, um die Aktivierung von SOCE in Monolayer-Zellen, suspendierten Zellen und Muskelfasern 5,8-10 messen. Die am häufigsten von dieser Fluoreszenz-Methoden verwendet, ist die direkte Überwachung der Dynamik der intrazellulären Ca 2 + aus dem Verhältnis von F und F 340 nm 380 nm (510 nm für die Emissions-Wellenlänge) des ratiometrische Ca 2 +-Indikator Fura-2. Um die Aktivität von unidirektionalen SOCE von intrazellulärer Ca 2 +-Freisetzung und Ca 2 +-Extrusion zu isolieren, wird ein Mn 2 +-Quenching-Assay häufig verwendet. Mn 2 + ist bekannt, können in die Zellen über SOCE Permeat während es undurchlässig für die Oberflächenmembran Extrusionsverfahren oder ER Aufnahme von Ca 2 + Pumpen aufgrund seiner sehr hohen Affinität nämlichh Fura-2. Als Ergebnis stellt das Abschrecken Fura-2 Fluoreszenz durch den Eintrag von extrazellulärem Mn 2 + in den Zellen induziert eine Messung der Aktivität von SOCE 9. Quotientenmeßschaltung und die Mn +2 Abschrecken Assays können auf einer Küvette-basierten Spektrofluorometer in einer Zellpopulation Modus oder in einem Mikroskop-System, um einzelne Zellen zu visualisieren durchgeführt werden. Der Vorteil einer einzigen Zelle Messungen ist, dass einzelne Zellen unterzogen, um Gen-Manipulationen ausgewählt werden unter Verwendung von GFP oder RFP-Reporter, so dass Studien in genetisch modifizierten oder mutierten Zellen werden. Diese räumliche Merkmale SOCE strukturell spezialisierten Skelettmuskel in Haut Muskelfasern durch gleichzeitiges Überwachen der Fluoreszenz von zwei geringe Affinität Ca 2 +-Indikatoren erreicht werden und gezielt auf die Abteile der Muskelfaser, wie zB Fluo-5N in der SR und Rhod- 5N im transversalen Tubuli 9,11,12.

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

Das Ausmaß der store-betriebenen Ca<sup&gt; 2 +</sup&gt; Eintrag (SOCE) kann überwacht werden, unter Verwendung von fluoreszierenden Ca<sup&gt; 2 +</sup&gt; Indikatoren. Mn<sup&gt; 2 +</sup&gt; Löschung solcher Indikatoren Assays SOCE in kultivierten Zellen und Fasern der Skelettmuskulatur. Eine Technik, so dass räumliche und zeitliche Auflösung von SOCE durch konfokale Bildgebung von mechanisch gehäutet Muskelfasern wird ebenfalls beschrieben.

Fluoreszenz-basierte Messung von Calcium-Shop-Eintrag in lebenden Zellen: aus kultivierten Krebszelle zu Skelettmuskelfaser

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 Aufrufe.  Robert Wood Johnson Medical School. Das Ausmaß der store-betriebenen Ca 2 + Eintrag (SOCE) kann überwacht werden, unter Verwendung von fluoreszierenden Ca 2 + Indikatoren. Mn 2 + Löschung solcher Indikatoren Assays SOCE in kultivierten Zellen und Fasern der Skelettmuskulatur. Eine Technik, so dass räumliche und zeitliche Auflösung von SOCE durch konfokale Bildgebung von mechanisch gehäutet Muskelfasern wird ebenfalls beschrieben.

Serie: 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.

Mitochondriale Isolation von der Skelettmuskulatur

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

Forschung

Biologie

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.

Reinigung von Vorläufern aus Skelettmuskeln

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.

Isolation und Kultur der einzelnen Muskelfasern und ihre Satelliten aus adulten Skelettmuskel

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.

Bewertung von Calcium in Sparks Intact Skelettmuskelfasern

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.

Isolierung von Blutgefäß abgeleiteten multi Vorläufer von menschlichen Skelettmuskel

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.

Die Messung von Calcium Schwankungen innerhalb des Retikulum von kultivierten glatten Muskelzellen Mit FRET-basierten konfokale Imaging

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.

Simultane Messung von Mitochondriale Calcium und mitochondrialen Membranpotentials in lebenden Zellen durch Fluoreszenzmikroskopie

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.

Live Cell Imaging von Pilzzellen Modi des Eintrags und subzelluläre Lokalisation der antimykotische Pflanze Defensine zu untersuchen

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.

Isolierung von hochwertigen atrialen und ventrikulären Myozyten der Maus für die simultane Messung von Ca2+ Transienten und L-Typ Calciumstrom

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.

Live-Bildgebung der Mikrotubulidynamik in Glioblastomzellen, die in das Zebrafischgehirn eindringen

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