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

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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><tbody><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (high glucose, pyruvate)</td><td>Life technologies</td><td>11995-065</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Matrigel Basement Membrane Matrix Growth Factor Reduced, 5 ml</td><td>Corning</td><td>356230</td><td>Keep at -20 &amp;deg;C until right before use</td></tr><tr><td>Newborn Calf Serum</td><td>Sigma-Aldrich</td><td></td><td>Keep at -20 &amp;deg;C until right before use</td></tr><tr><td>35 mm glass-bottomed plates</td><td>MatTek Corporation</td><td>P35G-1.5-14-C</td><td></td></tr><tr><td>250 ml 0.2 &amp;mu;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 &amp;deg;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 &amp;deg;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

Calcium Imaging of Cortical Neurons using Fura-2 AM

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths.  The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding.  Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.

סידן הדמיה של נוירונים קליפתיים באמצעות Fura, 02:00

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium ...

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.

Here, we describe a methodology to obtain a pure population of human myoblasts from adult muscle tissue. These cells are used to study in vitro skeletal muscle differentiation and, in particular, to study proteins involved in Ca2+ signaling.

בידוד של Myoblasts האדם, הערכה של בידול Myogenic, ו-מופעלות סידן מופעל מדידה

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for ...

Developmental Biology

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+ ...

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 ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

מדידת Fluxes סידן מהיר ב cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

מדידות סידן cytosolic בתאי האפיתל כליות על ידי cytometry הזרימה

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

ההדמיה המקומית Ca<sup&gt; 2 +</sup&gt; אותות בתאי יונקים בתרבית

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

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 ...

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of intracellular calcium release are the major intracellular calcium storage organelles, the endoplasmic reticulum (ER) and the more specialized sarcoplasmic reticulum (SR) in muscle cells. The dynamic release of calcium from these organelles is mediated by the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) with refilling occurring through the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. A genetically encoded calcium sensor (GECI) called CatchER was created to monitor the rapid calcium release from the ER/SR. Here, the detailed protocols for the transfection and expression of the improved, ER/SR-targeted GECI CatchER+ in HEK293 and C2C12 cells and its application in monitoring IP3R, RyR, and SERCA pump-mediated calcium transients in HEK293 cells using fluorescence microscopy is outlined. The receptor agonist or inhibitor of choice is dispersed in the chamber solution and the intensity changes are recorded in real time. With this method, a decrease in ER calcium is seen with RyR activation with 4-chloro-m-cresol (4-cmc), the indirect activation of IP3R with adenosine triphosphate (ATP), and inhibition of the SERCA pump with cyclopiazonic acid (CPA). We also discuss protocols for determining the in situ Kd and quantifying basal [Ca2+] in C2C12 cells. In summary, these protocols, used in conjunction with CatchER+, can elicit receptor mediated calcium release from the ER with future application in studying ER/SR calcium related pathologies.

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

We present protocols for the application of our targeted genetically-encoded calcium indicator (GECI) CatchER+ for monitoring rapid calcium transients in the endoplasmic/sarcoplasmic reticulum (ER/SR) of HEK293 and skeletal muscle C2C12 cells using real-time fluorescence microscopy. A protocol for the in situ Kd measurement and calibration is also discussed.

ניטור ER / SR סידן שחרור עם CA ממוקד<sup&gt; 2+</sup&gt; חיישן Catcher<sup&gt; +</sup

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of ...

<meta charset="utf8"></head><body>הדמיית סידן חד-תאי למחקרי הפעלת ערוצים

Single-Cell Calcium Imaging for Studying the Activation of Calcium Ion Channels

Single cell Ca2+ imaging is essential for the study of Ca2+ channels activated by various stimulations like temperature, voltage, native compound and chemicals et al. It primarily relies on microscopy imaging technology and the related Ca2+ indicator Fura-2/AM (AM is the abbreviation for Acetoxymethyl ester). Inside the cells, Fura-2/AM is hydrolyzed by esterases into Fura-2, which can reversibly bind with free cytoplasmic Ca2+. The maximum excitation wavelength shifts from 380nm to 340nm (when saturated with Ca2+) upon binding. The emitted fluorescence intensity is quantitatively related to the concentration of bound Ca2+. By measuring the 340/380 ratio, the Ca2+ concentration in the cytoplasm can be determined, eliminating errors caused by variations in the loading efficiency of the fluorescent probe among different samples. This technology allows for real-time, quantitative, and simultaneous monitoring of Ca2+ changes in multiple cells. The results are stored in &#8220;.XLSX&#8221; format for subsequent analysis, which is fast and generates intuitive change curves, greatly improving the detection efficiency. From different experimental perspectives, this article lists the use of this technology to detect Ca2+ signals in cells with endogenous or overexpressed channel proteins. Meantime, different methods for activating cells were also showed and compared. The aim is to provide readers with a clearer understanding of the usage and applications of single cell Ca2+ imaging.

<meta charset="utf8"></head><body>דימות סידן חד-תאי לחקר הפעלת תעלות יוני סידן

This article presents a method for real-time, quantitative monitoring of calcium ion (Ca2+) concentrations in cells using single-cell Ca2+ imaging with the Fura-2/AM dye. This technique enables efficient dye loading and accurate calculation of Ca2+ levels through fluorescence intensity ratios, making it a simple and rapid approach for research applications.

דימות סידן חד-תאי לחקר הפעלת תעלות יוני סידן

Single cell Ca2+ imaging is essential for the study of Ca2+ channels activated by various stimulations like temperature, voltage, native compound and ...

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

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סידן הדמיה של נוירונים קליפתיים באמצעות Fura, 02:00

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מיקוד Ca^{2 +} איתור חלוף של שריר חלק

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

מדידת Fluxes סידן מהיר ב cardiomyocytes

מדידות סידן cytosolic בתאי האפיתל כליות על ידי cytometry הזרימה

ההדמיה המקומית Ca

ניטור הומאוסטזה סידן endoplasmic reticulum שימוש

ניטור ER / SR סידן שחרור עם CA ממוקד

דימות סידן חד-תאי לחקר הפעלת תעלות יוני סידן