Biz okuma ve bu yazının düzenleme için Dr Nuh Weisleder teşekkür ederim. Bu çalışma MB 0535555N ZP, XZ için Amerikan Kalp Derneği SDG2630086, ve Ulusal Sağlık Enstitüleri RC2AR058962-01 MB için Araştırma Fonları UMDNJ 62-09 Vakfı tarafından desteklenmiştir.

<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>

Çıkar çatışması ilan etti.

Ca + 2-bağlayıcı ve Ca2 + serbest Fura-2 uyarma dalga boyları sırasıyla, 340 nm ve 380 nm olmasına rağmen, Ca 2 için Fura-2 iyi oranı dinamik aralığı + konsantrasyonunun ölçülmesi belirli bir başka dalga boylarında oluşabilir mikroskop sistemi. Bu tür değişimler dalga boyunda genellikle farklı optik bileşenlerin ilavesi ile optik yol üzerinde değişikliklere bağlı bulunmaktadır. Bu çalışmada, Fura-2 için uyarma dalga Fura-2 floresans spektral analizi yaparak 350 nm ve 390 nm olarak belirlendi. Dahil olmak üzere çeşitli kaynaklardan arasında bir denge, gelen sitozolüne sonuçlar intraselüler Ca2 + seviyesi intraselüler Ca2 + serbest bırakma, hücre dışı Ca2 + giriş yanı sıra, ER ve plazma membranında Ca2 + dışlama mekanizmasıdır. Hücre içi Ca 2 + sürümü ve Ca 2 + extr gelen tek yönlü SOC-aracılı Ca 2 + akını izole etmek içinusion, Mn 2 + soğutmadan test kullanılabilir. Mn 2 + SOCE yoluyla hücre içine nüfuz edebilmek için bilinen ama yüzey membran ekstrüzyon veya Ca 2 ER alımı + pompaları geçirmeyen edilir. Bu nedenle, flöresan söndürmesiyle SOCE aktivasyon derecesi tahmin hücrelerin içine tek yönlü Mn 2 + akısı bir ölçüm temsil eder. Mn 2 + soğutmadan deneyi gibi dalga boyunda, isosbestic noktada gerçekleştirilir Fura-2 floresans yoğunluğunu Ca2 + konsantrasyonu bağımsızdır. Edilir Her sistem için isosbestic noktası spektrum taraması ile tespit edilir. Alternatif olarak, Mn 2 + soğutmadan son değere Ca2 + konsantrasyonu 13 bağımsız şekilde herhangi iki dalga boyunda ayarlanmış floresans tarafından kaydedilebilir. Iskelet kaslarında SOCE faaliyet zamansal ve mekansal bilgi almak için, simultane ölçüm tekni sağlayan bir çift boya yöntemi, istihdamSOCE aktivite ve SR Ca 2 tenli yetişkin memelilerin EDL kas liflerinin + içerik nt. SR Ca 2 değişimler arasındaki ilişki + içeriği ve SOCE aktivite SR Ca 2 tükenmesi + depolama SOCE aktivasyon eşiği ve duyarlılık göstermek için çizilebilir. TT-yükleme çözüm ek Ca 2 T-tubul yüklemek için optimize edilmiştir + ve T-tubul ve SR-yükleme çözüm priming için maksimal SR Ca 2 + yükleme teşvik etmek ve ayrıca T-tubul yüklemek için tasarlanmıştır ~ 500 uM Ca 2 ile +. FCCP TT / SR-yükleme çözümü ve mitokondri gelen etkileri ortadan kaldırmak için SR-tüketen bir çözüm bulunur.

Özel reaktifler ve ekipman Tablo: <table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </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> Glass Bottom Bulaşık </td><td> MatTek Corp </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&#39;de, DMSO içinde çözülmüş ışıktan korunan conta sızdırmaz, </td></tr><tr><td> Fluo-5N AM </td><td> Invitrogen (Molecular Probes) </td><td> F14204 </td><td> -20 ° C, mühür sıkı, l korunmaktadıright </td></tr><tr><td> Rhod-5N </td><td> Invitrogen (Molecular Probes) </td><td> R14207 </td><td> -20 ° C, mühür sıkı ve ışıktan korunmalıdır </td></tr><tr><td> Kuvars Küvet </td><td> Starna Hücreleri </td><td> 3-S-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-toluen sülfonamid (BTS) </td><td> Sigma-Aldrich </td><td> 435600 </td><td></td></tr><tr><td> Kollajenaz Tip I </td><td> Sigma </td><td> C0130 </td><td></td></tr></tbody></table> Ekipman: <ol><li> Bir spectrofluorometer (Photon Technology International, NJ): ksenon ark lambası, bilgisayar kontrollü yüksek hızlı rastgele erişim monokromatör, küvet tabanlı emisyon monokromatör, Nikon TE-200 ters floresans MikroskopE, S Fluor 40x/1.30 yağlı objektif, PMT dedektörleri. </li><li> Argon lazer 453/477/488/514, HeNe lazer 543nm, Nikon TE2000 inverted mikroskop, 60X objektif (NA 1.4, yağ): Lazer Konfokal Mikroskobu (BioRad Radiance2100) tarama. </li><li> Bir büyütme gücü&gt; 100 (Zeiss STEMI SV11 Apo) ile bir stereomikroskop. </li></ol>

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

Mağaza önceki kapasitif Ca 2 + girişi olarak adlandırılan, Ca 2 + girişi (SOCE) ameliyat, + tükenmiş endoplazmik retikulum (ER) veya sarkoplazmik retikulum (SR) Ca 2 + depolarının doldurulmasıdır hücre içine ekstraselüler Ca 2 akını için sıkı regüle mekanizmadır 1,2. Ca 2 + bir yerde ikinci bir haberci olduğu için, o SOCE çoğalması, apoptoz, gen transkripsiyonu ve motilite dahil, hücresel süreçlerin çeşitli önemli rol oynar görmek şaşırtıcı değildir. Epitel hücreleri ve iskelet kasları dahil olmak üzere hemen hemen tüm hücre tipleri, içinde geniş görülmesi nedeniyle, bu yolu büyük ilgi 3,4 aldı. Ancak, farklı hücre tipleri ve fizyolojik fonksiyonu SOCE özellikleri heterojenite hala 5-7 net değil. SOCE fonksiyonel kanal özellikleri thi hakkında bilgi büyük bir gövde ise, yama klemp çalışmalarla ortaya edilebilirs yolu çünkü yüksek verimli tarama rahatlığının ve fizibilite floresans tabanlı hücre içi Ca 2 + ölçümleri ile elde edilmiştir. Bu raporun amacı, tek tabakalı hücreler, askıya hücreleri ve kas lifleri 5,8-10 içinde SOCE aktivasyonu ölçmek için bir kaç floresans tabanlı yöntemler özetlemektir. En yaygın olarak floresans bu yöntem kullanılır, doğrudan intraselüler Ca2 + dinamiği izlemek için F ve 340 nm oranlı metrik Ca2 + göstergesi Fura-2 F 380nm (emisyon dalga boyu için 510 nm) oranı kullanılarak bir. Hücre içi Ca2 + serbest bırakılması ve Ca arasından 2 yönlü SOCE aktivitesini izole etmek için + ekstrüzyon, bir Mn 2 + soğutmadan assay sıkça kullanılır. Mn 2 + o yüzeye membran ekstrüzyon süreçlere veya Ca 2 tarafından ER uptake geçirmez iken SOCE yoluyla hücre içine nüfuz edebilir olduğu bilinmektedir + olan çok yüksek bir afiniteye wit nedeniyle pompalarh Fura-2. Bunun sonucu olarak, + hücrelerine ekstraselüler Mn 2 girişi ile indüklenen Fura-2 floresans soğutmadan SOCE 9 faaliyeti, bir ölçüm temsil eder. Oranlı metrik ölçümü ve Mn +2 soğutmadan deneyleri, bir hücre popülasyonunu modunda ya da tek hücrelerin canlandırmak için mikroskop tabanlı bir sistem içerisinde bir küvet bazlı spectrofluorometer üzerinde gerçekleştirilebilir. Tek hücre ölçümlerin avantajı gen manipülasyonu tabi tek tek hücrelerin genetik olarak modifiye edilmiş ya da mutasyon hücrelerinde çalışmaları sağlayan GFP veya RFP gazetecilere kullanılarak seçilebilir olmasıdır. Yapısal olarak özel bir iskelet kasında SOCE arasında zamanmekansal özellikleri aynı anda iki düşük afinite Ca 2 floresans izlenerek kabuklu kas fiberler elde edilebilir SR ve bu gibi flüoresan 5N olarak kas fiberin özel bir bölme, hedeflenen + göstergeleri Rhod- transvers tübüller 9,11,12 olarak 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

Mağaza çalışan Ca ölçüde<sup&gt; 2 +</sup&gt; Giriş (SOCE) floresan Ca kullanılarak izlenebilir<sup&gt; 2 +</sup&gt; Göstergeleri. Mn<sup&gt; 2 +</supKültür hücreleri ve kas lifleri bu tür göstergeler testler SOCE bir&gt; su verme. Mekanik tenli kas liflerinin konfokal görüntüleme ile SOCE uzaysal ve zamansal çözünürlüğe izin veren bir tekniği de tarif edilir.

Canlı hücreler Mağaza çalışan Kalsiyum Giriş Floresans tabanlı Ölçümü: Kültür Kanseri Hücre gelen İskelet Kası Fiber

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 Görüntüleme.  Robert Wood Johnson Medical School. Mağaza çalışan Ca ölçüde 2 + Giriş (SOCE) floresan Ca kullanılarak izlenebilir 2 + Göstergeleri. Mn 2 + su verme. Mekanik tenli kas liflerinin konfokal görüntüleme ile SOCE uzaysal ve zamansal çözünürlüğe izin veren bir tekniği de tarif edilir.

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.

İskelet Kası Mitokondrial İzolasyon

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

Biyoloji

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.

İskelet Kası progenitörlerin saflaştırılması

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.

Yetişkin İskelet Kası gelen bireysel Myofibers ve Uydu Hücre İzolasyonu ve Kültür

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.

Kalsiyum değerlendirilmesi Bozulmamış İskelet kas liflerinin içinde Sparks

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.

İnsan İskelet Kası Kan-damar-türevli Multipotent Öncüleri İzolasyonu

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.

Kullanımı Kültür düz kas hücrelerinin sarkoplazmik retikulum içinde Kalsiyum Değişiminin Ölçülmesi FRET tabanlı Konfokal Görüntüleme

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.

Floresan Mikroskopi Canlı hücreler Potansiyel Mitokondriyal Kalsiyum ve Mitokondriyal Membran Eşzamanlı Ölçümü

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.

Canlı hücre görüntüleme modları giriş ve Antifungal bitki Defensins hücre altı yerelleştirme araştırmak için mantar hücrelerinin

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+ geçici ve L-tipi kalsiyum akımının eşzamanlı ölçümleri için yüksek kaliteli murin atriyal ve ventriküler miyositlerin izolasyonu

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

Mitokondriyal Membran Potansiyeli ve Süperoksit Seviyelerinin Miktar Tayini

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.

Yazar Gündemi: HeLa Hücrelerinde Canlı Görüntüleme Kullanılarak Mitokondriyal Membran Potansiyelinin ve Süperoksit Seviyelerinin Floresan Tabanlı Miktar Tayini  

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.

HeLa hücrelerinde canlı görüntüleme kullanılarak mitokondriyal membran potansiyelinin ve süperoksit seviyelerinin floresan bazlı ölçümü

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