Ringraziamo il dottor Noah Weisleder per la lettura e la modifica di questo manoscritto. Questo lavoro è stato supportato da Research Grants UMDNJ Foundation 62-09 a ZP, American Heart Association SDG2630086 a XZ, 0535555N a MB, e la National Institutes of Health RC2AR058962-01 a MB.

<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>

Non ci sono conflitti di interesse dichiarati.

Sebbene le lunghezze d&#39;onda di eccitazione di Ca 2 +-binding e Ca 2 + libero Fura-2, 340 nm e 380 nm, rispettivamente, la gamma dinamica migliore rapporto di Fura-2 per la concentrazione di Ca 2 + misura può avvenire in altre lunghezze d&#39;onda in un particolare microscopio sistema. Tali variazioni di lunghezza d&#39;onda di solito sono dovute a variazioni del percorso ottico con l&#39;aggiunta di vari componenti ottici. In questo studio, le lunghezze d&#39;onda di eccitazione di Fura-2 sono stati determinati a 350 nm e 390 nm effettuando l&#39;analisi spettrale di Fura-2 fluorescenza. Il livello intracellulare Ca 2 + nei risultati citosol di una bilancia di fonti diverse, comprese intracellulare Ca 2 + rilascio, Ca 2 + extracellulare ingresso, così come Ca 2 + meccanismo di esclusione a livello della membrana plasmatica e ER. Per isolare il unidirezionale SOC-mediata Ca 2 + intracellulare afflusso di Ca 2 + release e Ca 2 + extrperfusione, l&#39;Mn 2 + quenching saggio può essere utilizzato. Mn 2 + è noto per essere in grado di permeare nelle cellule tramite Soče ma è impermeabile alla superficie della membrana estrusione o assorbimento ER da Ca 2 + pompe. Pertanto, estinzione di fluorescenza rappresenta una misura di unidirezionale Mn 2 + flusso in cellule che stima il grado di attivazione di Soče. Mn 2 + bonifica saggio viene eseguito al punto isosbestic, a tale lunghezza d&#39;onda Fura-2 intensità di fluorescenza è indipendente dalla concentrazione di Ca 2 +. Il punto isosbestic per ogni sistema dovrebbe essere determinata mediante scansione spettro. In alternativa, Mn 2 + bonifica può essere registrato da fluorescenza regolata in qualsiasi due lunghezze d&#39;onda in modo tale che il valore finale è indipendente concentrazione di Ca 2 + 13. Per ottenere le informazioni spaziali e temporale di attività di Soče nei muscoli scheletrici, abbiamo impiegato un processore dual-colorante metodo che consenta MeasureMe simultaneant di attività Soče e SR Ca 2 + contenuto in pelle dei mammiferi adulti fibre muscolari EDL. La correlazione tra i cambiamenti nella SR Ca 2 + attività Soče contenuti e possono essere tracciate per indicare la soglia e la sensibilità di attivazione Soče alla deplezione del Ca + SR 2 di stoccaggio. La TT-loading soluzione è ottimizzata per caricare ulteriormente le T-tubuli con Ca 2 + e per l&#39;innesco dei T-tubuli e la SR-loading soluzione è progettata per promuovere la massima SR Ca 2 + carico e per caricare ulteriormente le T-tubuli con ~ 500 pM Ca 2 +. FCCP è incluso nel TT / SR-caricamento soluzione e il SR deplezione soluzione per eliminare gli effetti della mitocondri.

Tabella di reagenti e attrezzature specifiche: <table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> RPMI 1640 </td><td> Mediatech </td><td> 10-040-CV </td><td></td></tr><tr><td> Ham F-12 </td><td> Mediatech </td><td> 10-080-CV </td><td></td></tr><tr><td> DMEM </td><td> Mediatech </td><td> 10-013-CV </td><td></td></tr><tr><td> Fondo di Vetro 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, chiusura ermetica, al riparo dalla luce, dissolto in DMSO </td></tr><tr><td> Fluo-5N AM </td><td> Invitrogen (Molecular Probes) </td><td> F14204 </td><td> -20 ° C, tenuta, protetto dal light </td></tr><tr><td> Rhod-5N </td><td> Invitrogen (Molecular Probes) </td><td> R14207 </td><td> -20 ° C, chiusura ermetica, al riparo dalla luce </td></tr><tr><td> Quarzo Cuvette </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-benzil-p-toluene solfonammide (BTS) </td><td> Sigma-Aldrich </td><td> 435600 </td><td></td></tr><tr><td> Collagenasi di tipo I </td><td> Sigma </td><td> C0130 </td><td></td></tr></tbody></table> Attrezzatura: <ol><li> A spettrofluorimetro (Photon Technology International, NJ): lampada allo xenon ad arco, controllato dal computer ad alta velocità monocromatore ad accesso casuale, cuvette-based emissioni monocromatore, Nikon TE-200 a fluorescenza invertito microscope, S Fluor 40x/1.30 obiettivo di olio, rivelatori di PMT. </li><li> Microscopio confocale a scansione laser (BioRad Radiance2100): argon laser 453/477/488/514, HeNe laser 543nm, Nikon TE2000 microscopio invertito, obiettivo 60X (NA 1.4, olio). </li><li> A stereomicroscopio con un potere di ingrandimento&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

Conservare operato Ca 2 + voce (Soče), in precedenza chiamato capacitivo Ca 2 + voce, è un meccanismo strettamente regolata per influsso di Ca 2 + extracellulare nelle cellule per ricostituire impoverito reticolo endoplasmatico (ER) o reticolo sarcoplasmatico (SR) Ca 2 + negozi 1,2. Dal Ca 2 + è un messaggero ubiquitario secondo, non è sorprendente vedere che Soče svolge un ruolo importante in una varietà di processi cellulari, compresa la proliferazione, l&#39;apoptosi, la trascrizione del gene e la motilità. Grazie alla sua ampia presenza in quasi tutti i tipi cellulari, comprese le cellule epiteliali ed i muscoli scheletrici, questo percorso ha ricevuto grande interesse 3,4. Tuttavia, l&#39;eterogeneità delle caratteristiche Soče in diversi tipi di cellule e la funzione fisiologica non sono ancora chiari 5-7. Le proprietà funzionali dei canali di Soče può essere rivelato da patch-clamp studi, mentre un ampio corpus di conoscenza thivia s è stata acquisita dalla basato sulla fluorescenza intracellulare di Ca 2 + misure a causa della sua convenienza e fattibilità per high-throughput screening. L&#39;obiettivo di questa relazione è quello di riassumere alcuni fluorescenza a base di metodi per misurare l&#39;attivazione di Soče nelle cellule monostrato, cellule in sospensione e fibre muscolari 5,8-10. Il più comunemente usato di questi metodi fluorescenza è di controllare direttamente le dinamiche di Ca 2 + intracellulare utilizzando il rapporto di F 340nm e 380nm F (510 nm di lunghezza d&#39;onda di emissione) del raziometrico Ca 2 + indicatore Fura-2. Per isolare l&#39;attività di Soče unidirezionale dal intracellulare Ca 2 + release e Ca 2 + di estrusione, una tempra Mn 2 + test è usato frequentemente. Mn 2 + è noto per essere in grado di permeare nelle cellule tramite Soče mentre è impermeabile ai processi superficie della membrana estrusione o per assorbimento da ER Ca 2 + pompe a causa del suo ingegno un&#39;elevata affinitàh Fura-2. Come risultato, la tempra di Fura-2 fluorescenza indotta dalla entrata di Mn 2 + extracellulare in cellule rappresenta una misura dell&#39;attività di Soče 9. Raziometrica misurazione e le Mn +2 saggi di spegnimento può essere eseguita su una cuvetta basato spettrofluorimetro in una modalità popolazione cellulare o in un microscopio sistema basato su visualizzare le cellule singole. Il vantaggio delle misurazioni singola cellula è che le cellule individuo soggetto a manipolazioni genetiche possono essere selezionati tramite GFP o reporter RFP, che consente studi in cellule geneticamente modificate o mutato. Le caratteristiche spaziotemporali di Soče in strutturalmente specializzato muscolo scheletrico può essere realizzato in fibre muscolari pelle contemporaneamente monitorando la fluorescenza dei due bassa affinità di Ca 2 + indicatori mirati a specifici compartimenti della fibra muscolare, come Fluo-5N nella SR e di Rhod 5N nella trasversale 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

L&#39;entità del negozio ad azionamento Ca<sup&gt; 2 +</supIngresso&gt; (Soče) possono essere monitorati con fluorescente Ca<sup&gt; 2 +</sup&gt; Indicatori. Mn<sup&gt; 2 +</sup&gt; Tempra di tale Soče indicatori saggi in cellule in coltura e delle fibre muscolari scheletriche. Una tecnica che permette la risoluzione spaziale e temporale delle Soče da confocale delle fibre muscolari meccanicamente dalla pelle viene anche descritto.

Basato sulla fluorescenza Misura Store-Entry operato di calcio in cellule vive: dal Cancer Cell coltura per fibra muscolare scheletrica

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

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Research

JoVE Journal

Biology

21.1K Views.  Robert Wood Johnson Medical School. L'entità del negozio ad azionamento Ca 2 + (Soče) possono essere monitorati con fluorescente Ca 2 + Indicatori. Mn 2 + Tempra di tale Soče indicatori saggi in cellule in coltura e delle fibre muscolari scheletriche. Una tecnica che permette la risoluzione spaziale e temporale delle Soče da confocale delle fibre muscolari meccanicamente dalla pelle viene anche descritto.

Video: 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.

Isolamento dei mitocondri dal muscolo scheletrico

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

Ricerca

Biologia

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.

Purificazione di progenitori dal muscolo scheletrico

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.

Isolamento e la coltura di miofibre individuali e delle loro cellule satelliti da adulto muscolo scheletrico

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.

Valutazione del calcio Sparks in intatte fibre muscolari scheletriche

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.

Isolamento dei vasi sanguigni derivati ​​da Multipotent precursori del muscolo scheletrico umano

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.

Misura di fluttuazioni calcio all&#39;interno del reticolo sarcoplasmatico di coltura cellule muscolari lisce Uso FRET-based Imaging confocale

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.

Misurazione simultanea di mitocondriale calcio e mitocondriale potenziale di membrana in cellule vive di Microscopia fluorescente

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.

Imaging di cellule vive di cellule fungine per studiare modalità di entrata e localizzazione subcellulare delle defensine antifungino pianta

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.

Isolamento di miociti atriali e ventricolari murini di alta qualità per misurazioni simultanee di transitori Ca2+ e corrente di calcio di tipo L

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

Quantificazione del potenziale di membrana mitocondriale e dei livelli di superossido

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, various mitochondrial quality control mechanisms cooperate to maintain a healthy mitochondrial network. One such pathway is mitophagy, where PTEN-induced kinase 1 (PINK1) and Parkin phospho-ubiquitination of damaged mitochondria facilitate autophagosome sequestration and subsequent removal from the cell via lysosome fusion. Mitophagy is important for cellular homeostasis, and mutations in Parkin are linked to Parkinson's disease (PD). Due to these findings, there has been a significant emphasis on investigating mitochondrial damage and turnover to understand the molecular mechanisms and dynamics of mitochondrial quality control. Here, live-cell imaging was used to visualize the mitochondrial network of HeLa cells, to quantify the mitochondrial membrane potential and superoxide levels following treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. In addition, a PD-linked mutation of Parkin (ParkinT240R) that inhibits Parkin-dependent mitophagy was expressed to determine how mutant expression impacts the mitochondrial network compared to cells expressing wild-type Parkin. The protocol outlined here describes a simple workflow using fluorescence-based approaches to quantify mitochondrial membrane potential and superoxide levels effectively.

Autore in primo piano: Quantificazione basata sulla fluorescenza del potenziale di membrana mitocondriale e dei livelli di superossido utilizzando l'imaging dal vivo nelle cellule HeLa  

This technique describes an effective workflow to visualize and quantitatively measure mitochondrial membrane potential and superoxide levels within HeLa cells using fluorescence-based live imaging.

Quantificazione basata sulla fluorescenza del potenziale di membrana mitocondriale e dei livelli di superossido utilizzando l'imaging dal vivo nelle cellule HeLa

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, ...