עבודה זו נתמכה על ידי מימון מן המכון הקנדי לבריאות מחקר [MOP-1112666 כדי CVB &amp; ME].

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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+calcium+and+cyclic+AMP+in+cell+proliferation.">The roles of calcium and cyclic AMP in cell proliferation.</a> Ann N Y Acad Sci. 339 (1), 216-240 (1980).</li><li>Nicotera, P., Orrenius, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+calcium+in+apoptosis.">The role of calcium in apoptosis.</a> Cell Calcium. 23 (2-3), 173-180 (1998).</li><li>Wei, C., Wang, X., Chen, M., Ouyang, K., Song, L. S., Cheng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+flickers+steer+cell+migration.">Calcium flickers steer cell migration.</a> Nature. 457 (7231), 901-905 (2009).</li><li>Ashby, M. C., Tepikin, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER+calcium+and+the+functions+of+intracellular+organelles.">ER calcium and the functions of intracellular organelles.</a> Sem Cell Dev Biol. 12 (1), 11-17 (2001).</li><li>Mattson, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER+calcium+and+Alzheimer's+disease:+in+a+state+of+flux.">ER calcium and Alzheimer's disease: in a state of flux.</a> Sci signal. 3 (114), (2010).</li><li>Rutkowski, D. T., Kaufman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+trip+to+the+ER:+coping+with+stress.">A trip to the ER: coping with stress.</a> Trends Cell Biol. 14 (1), (2004).</li><li>Schroder, M., Kaufman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER+stress+and+the+unfolded+protein+response.">ER stress and the unfolded protein response.</a> Mutat Res. 569 (1-2), 29-63 (2005).</li><li>Palmer, A. E., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+calcium+signaling+using+genetically+targetable+fluorescent+indicators.">Measuring calcium signaling using genetically targetable fluorescent indicators.</a> Nat Protoc. 1 (3), 1057-1065 (2006).</li><li>Poburko, D., Liao, C. H., van Breemen, C., Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+regulation+of+sarcoplasmic+reticulum+Ca2++content+in+vascular+smooth+muscle+cells.">Mitochondrial regulation of sarcoplasmic reticulum Ca2+ content in vascular smooth muscle cells.</a> Circ Res. 104 (1), 104-112 (2009).</li><li>Esfandiarei, M., Fameli, N., Choi, Y. Y., Tehrani, A. Y., Hoskins, J. G., van Breemen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Waves+of+calcium+depletion+in+the+sarcoplasmic+reticulum+of+vascular+smooth+muscle+cells:+an+inside+view+of+spatiotemporal+Ca2++regulation.">Waves of calcium depletion in the sarcoplasmic reticulum of vascular smooth muscle cells: an inside view of spatiotemporal Ca2+ regulation.</a> PloS one. 8 (2), e55333 (2013).</li><li>Cobbold, P. H., Rink, 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=Fluorescence+and+bioluminescence+measurement+of+cytoplasmic+free+calcium.">Fluorescence and bioluminescence measurement of cytoplasmic free calcium.</a> Biochem J. 248 (2), 313-328 (1987).</li><li>Park, M. K., Ashby, M. C., Erdemli, G., Petersen, O. H., Tepikin, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perinuclear,+perigranular+and+sub-plasmalemmal+mitochondria+have+distinct+functions+in+the+regulation+of+cellular+calcium+transport.">Perinuclear, perigranular and sub-plasmalemmal mitochondria have distinct functions in the regulation of cellular calcium transport.</a> The EMBO journal. 20 (8), 1863-1874 (2001).</li><li>Lovett, J. L., Sibley, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+calcium+stores+in+Toxoplasma+gondii+govern+invasion+of+host+cells.">Intracellular calcium stores in Toxoplasma gondii govern invasion of host cells.</a> J Cell Sci. 116 (14), 3009-3016 (2003).</li><li>Kleinman, H. K., Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+basement+membrane+matrix+with+biological+activity.">Matrigel: basement membrane matrix with biological activity.</a> Semin Cancer Biol. 15 (5), 378-386 (2005).</li><li>Palmer, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++indicators+based+on+computationally+redesigned+calmodulin-peptide+pairs.">Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs.</a> Chem Biol. 13 (5), 521-530 (2006).</li></ol>

החוקרים מצהירים כי אין להם אינטרסים כלכליים מתחרים.

פרוטוקול זה מתאר את transfection של VSMCs עם אדנו D1SR ללמוד Ca 2 + ריכוזים בתוך לומן של SR. שיטה זו מאפשרת מדידה ישירה של Ca 2 + בתוך אברון זה, כמו גם את התנועה לתוך / מתוך SR כתוצאה היישום של סוכנים שונים נע בסידן. לשיטה זו יתרונות רבים עבור חוקרים עובדים לקראת הבנה מקיפה של ?...

Ca 2 + הוא יון חשוב מאוד למצוא למכביר בתוך כל תא ותא בגוף. יש לו תפקידים משמעותיים רבים תפקודים תאיים שונים, לרבות גידול, התפשטות, הגירה, ו אפופטוזיס 1-4. היא מבוססת היטב כי Ca 2 + יכול להשפיע על תהליכים אלה הם באמצעות פעולות ישירות ועקיפות, ולכן, שינויים לריכוז הנורמלי התאי של יון זה יכול לגרום בקלות תוצאות שליליות עבור תאים חולים. ערך SR נחשב החנות הגדולה התאי Ca 2 + בתוך התא 5. רמות מצב יציב של Ca 2+ הן SR ו הציטופלסמה נשמרות בדרך כלל באמצעות זרימה מתמדת לתוך ומתוך ערוצי -transporting Ca 2+ של אברון זה. התנאים המלחיצים המוטלים תאים בשל כל צורה של פציעה או מחלה הם נפוצים הקשורים שינויים משמעותיים SR Ca 2 + כי להמשיך להיות השפעות ארוכות טווח על שהואalth של התא. בשנת הקשה ביותר של המקרים, את חוסר היכולת של תא הדגיש להתאושש ולשמור Ca SR מצב יציב 2+ רמות אולי אפילו לשיאה למוות על ידי אפופטוזיס 6-8. מחקר נוכחי לתוך דינמיקת הסלולר Ca 2+ מוגבל על ידי העובדה כי בדיקת מחקרים מעטים אברון תוכן החנות 2 + Ca ישירות 9-11. המנהג הנפוץ ביותר במקום כרוך מדידת רמות ציטופלסמית Ca 2 + מדידות עקיפות של שינויים בתוכן SR Ca 2 + 12-14. בניסויים אלה, Ca 2 + מושרת נפוצה להשתחרר מן SR באמצעות סוכנים תרופתיים גורמים הדלדול של אברון (למשל thapsigargin). מסקנות אז נמשכו לגבי שינויי SR Ca 2 + ומתבססים על תנודות הריכוזים ציטופלסמית Ca 2+. למרות היכולת הנותרים לחוקרים להסיק מסקנות כאלה בתוך ma עקיפהnner, שיטה זו של מדידת 2+ SR Ca היא ללא ספק דרך עקיפה כדי ללקט מידע כזה, עם מגבלות רבות בנוגע לפרשנות של הנתונים שנאספו. כדי לעקוף מגבלה ברורה זו, יש צורך למדוד את כמות Ca 2 + מצאה ישירות בתוך הרשת לומינל SR. ויטל לתוצאה הסופית של יכולת להקליט רמות Ca 2+ SR intraluminal ישירות הם כלי תרבית תאים ומחוון Ca 2+ בשימוש. עבור הנתונים הפניה בכתב היד הנוכחית, חשוב לציין כי VSMCs בשימוש בא משושלת תא קפוא. תאים היו בתרבית הבינונית של הנשר השונה של Dulbecco (DMEM) + 10% בת-יומה בסרום (NCS) מעל הקטעים 22-26 עבור הניסויים שתוארו, וטופח על 37 מעלות צלזיוס קבוע עם 5% CO 2 אספקה. באמצעות השיטה הנוכחית, למשל, תאים כבר גדלו מאוד בהצלחה על תערובת חלבון המדמה את תאייםהסביבה של סוגים שונים של רקמות 15. חשוב להצלחה לאורך הוריד של SR Ca 2 + מחקר הוא סוג של תערובת חלבונים המשמשים; במקרה זה, מגוון גורם צמיחה נמוך היה נחוץ כדי למנוע מרכיבי הכלי הזה מאת משפיע על האיתות הרגילה Ca 2+ ותנועות המתרחשות כל זמן בתוך התאים הנבדקים. בעקבות גידול מוצלח של תאי בדיקה, מחוון Ca 2+ חייב גם להיות הציג ביעילות תאים בתרבית אלה. זה הפך להיות אפשרי באמצעות וקטור adenoviral שנושא את SR-המתגורר Ca 2 + אינדיקטור D1SR. כדי להשיג יעילות transfection גבוהה, התאים חייבים להיות מודגרות עם וקטורים ויראליים לפחות 36-48 שעות לפני הדמיה. הכנה זו מספקת כלי אמין למדידת Ca 2 + ארעיים בתוך לומן SR עם דיוק שחזור גבוה. מחוון D1SR להשתמש בפרוטוקול זה היא גרסה שונה של D1ER Ca 2 + indicator כי נוצרה במקור על ידי המעבדה של ד&quot;ר רוג&#39;ר צ&#39;יין באוניברסיטת קליפורניה, סן דייגו, 9,16 ארה&quot;ב. D1ER המקורי שייך לדור שני של אינדיקטורים מקודדים גנטי Ca 2+ שנקראו cameleons המציגים רגישויות 2+ Ca פני טווח רחב הרבה יותר (0.5-160 מיקרומטר) לעומת מדדים קודמים Ca 2 + 9,16. D1SR גרסה חדשה, מתנה סוג מד&quot;ר וויין חן (מאוניברסיטת אלברטה, קנדה), לעומת זאת, נושא רצף calsequestrin מוטציה (במקום רצף calreticulin כמו D1ER המקורי). Calsequestrin מוטציה יש מופחת מחייב Ca 2 + הפותר את הבעיה של מתחרים עם calsequestrin אנדוגני מחייב Ca 2 + בתוך SR לומן 11. מחוון D1SR נושאת חלבון פלואורסצנטי ציאן משופרת קטום (CFP) ו חלבון פלואורסצנטי צהוב (YFP) כי לאגד על ידי חלבון מקשר המכיל calmodulin שונה (CAM) ו M13 (tהוא 26-שאריות פפטיד של קינאז אור-שרשרת שרירן הנקשרת רצפים CAM). רצף CAM-M13 שונה כדי למנוע M13 מלהיקשר calmodulin אנדוגני. כמו כן, על מנת להבטיח שימור SR, רצף calsequestrin נוסף על קצה 5&#39;של CFP 11. כאשר חייב Ca 2 +, תחום CAM-M13 עובר שינויים מרחביים כי לגרום לעלייה בהעברת האנרגיה בין CFP האיגוף ו YFP, הנרשם כעלייה את עוצמת אות הסריג. מצד השני, כאשר הריכוז של SR לומינל Ca 2 + טיפות, תחום CAM-M13 עובר שינויים מרחביים הפוכים, וכתוצאה מכך ירידת העברת האנרגיה בין CFP האיגוף ו YFP, וירידה משמעותית בעוצמת אות סריג .

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (high glucose, pyruvate)</td>
			<td>Life technologies</td>
			<td>11995-065</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix Growth Factor Reduced, 5mL</td>
			<td>Corning</td>
			<td>356230</td>
			<td>Keep at -20&#176;C until right before use</td>
		</tr>
		<tr>
			<td>Newborn Calf Serum</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Keep at -20&#176;C until right before use</td>
		</tr>
		<tr>
			<td>35mm glass-bottomed plates</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL 0.2 &#956;M vented blue plug seal cap flask</td>
			<td>BD Falcon</td>
			<td>353136</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA Solution</td>
			<td>Life technologies</td>
			<td>25200056</td>
			<td>Keep at -20&#176;C until right before use</td>
		</tr>
		<tr>
			<td>50 mL Polypropylene Conical Tube</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>D1SR</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Gift</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8779</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo 4-AM</td>
			<td>Life technologies</td>
			<td>F-23917</td>
			<td>Keep at -20&#176;C until right before use</td>
		</tr>
		<tr>
			<td>1.5 mL Black Microcentrifuge Tubes</td>
			<td>Argos Technologies</td>
			<td>T7100BK</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica TCS SP5 confocal microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Application Suite 2.6.3 (with FRET SE Wizard application)</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 5.0</td>
			<td>GraphPad Software, Inc.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

סעיף זה מספק דוגמאות של התוצאות ניתן להשיג באמצעות השיטה המתוארת ללכוד SR לומינל Ca 2+ נתוני ריכוז, בהשוואה לאופן מועסק בדרך של בעקיפין מדידת שינויי אברון ריכוזי החנות 2 + Ca ידי התבוננות תנודות ציטופלסמית Ca 2+ . <p class="jove_content" fo:keep-together.w...

Watch this Scientific Journal Video about Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging at JoVE.com

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

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.

מדידת תנודות סידן בתוך הרטיקולום sarcoplasmic של בתאי שריר חלק בתרבית באמצעות מבוססי סריג הדמיה Confocal

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

measurement-calcium-fluctuations-within-sarcoplasmic-reticulum

Research

JoVE Journal

Biology

7.6K Views.  University of British Columbia. 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.

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

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

מיקוד Ca 2 + איתור חלוף של שריר חלק

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

בידוד של האדם עורקי טבורי תאים שרירים חלקים (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

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

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

הקרינה מבוסס מדידה הכניסה חנות המופעל הסידן בתאים חיים: מתא הסרטן תרבותי כדי סיב שריר שלד

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

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

בידוד של תאי שריר חלקים עורק ריאה מעכברים ילודים

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

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.

הערכה של סידן ניצוצות בסיבי שריר שלד שלמים

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

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

ויזואליזציה של תחומי משנה reticulum endoplasmic בתאים בתרבית

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

מדידת pH בו זמנית בendocytic ותאי Cytosolic בתאים חיים באמצעות מיקרוסקופיה confocal

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

מדידה של תפקוד של שריר חלק ברקמות מבודדות אמבט-היישומים למחקר פרמקולוגיה

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

מיפוי אופטי של Intra-sarcoplasmic reticulum Ca<sup&gt; 2 +</sup&gt; ופוטנציאל הטרנסממברני בלב ארנב Langendorff-perfused

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

ניטור הומאוסטזה סידן endoplasmic reticulum שימוש<em&gt; Gaussia</em&gt; לוציפראז SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

מדידת תנודות סידן בתוך הרטיקולום sarcoplasmic של בתאי שריר חלק בתרבית באמצעות מבוססי סריג הדמיה Confocal

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מיקוד Ca^{2 +} איתור חלוף של שריר חלק