עבודה זו נתמכה על ידי HL094536 (BJH).

<ol>
	<li>Kroemer, G., Galluzzi, L., Brenner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Membrane+Permeabilization+in+Cell+Death.">Mitochondrial Membrane Permeabilization in Cell Death.</a> Physiol. Rev. 87, 99-163 (2007).</li><li>Elrod, J., Wong, R., Mishra, S., Vagnozzi, R. J., Sakthievel, B., Goonasekera, S. A., Karch, J., Gabel, S., Farber, J., Force, T., Brown, J. H., Murphy, E., Molkentin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclophilin+D+controls+mitochondrial+pore-dependent+Ca2++exchange,+metabolic+flexibility,+and+propensity+for+heart+failure+in+mice.">Cyclophilin D controls mitochondrial pore-dependent Ca2+ exchange, metabolic flexibility, and propensity for heart failure in mice.</a> J. Clin. Invest. 120, 3680-3687 (2010).</li><li>Hom, J. R., Quintanilla, R. A., Hoffman, D. L., de Mesy Bentley, K. L., Molkentin, J. D., Sheu, S. S., Porter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+permeability+transition+pore+controls+cardiac+mitochondrial+maturation+and+myocyte.">The permeability transition pore controls cardiac mitochondrial maturation and myocyte.</a> 21, 469-478 (2011).</li><li>Halestrap, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+mitochondrial+permeability+transition+pore.">What is the mitochondrial permeability transition pore.</a> Journal of Molecular and Cellular Cardiology. 46, 821-831 (2009).</li><li>Wei, A. C., Liu, T., Cortassa, S., Winslow, R. L., O'Rourke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Ca2++influx+and+efflux+rates+in+guinea+pig+cardiac+mitochondria:+low+and+high+affinity+effects+of+cyclosporine+A.">Mitochondrial Ca2+ influx and efflux rates in guinea pig cardiac mitochondria: low and high affinity effects of cyclosporine A.</a> Biochim. Biophys. Acta. 1813, 1373-1381 (2011).</li><li>Saks, V. A., Kuznetsov, A. V., Kupriyanov, V. V., Miceli, M. V., Jacobus, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creatine+kinase+of+rat+heart+mitochondria.+The+demonstration+of+functional+coupling+to+oxidative+phosphorylation+in+an+inner+membrane-matrix+preparation.">Creatine kinase of rat heart mitochondria. The demonstration of functional coupling to oxidative phosphorylation in an inner membrane-matrix preparation.</a> J. Biol. Chem. 260, 7757-7764 (1985).</li><li>Boehm, E. A., Jones, B. E., Radda, G. K., Veech, R. L., Clarke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+uncoupling+proteins+and+decreased+efficiency+in+palmitate-perfused+hyperthyroid+rat+heart.">Increased uncoupling proteins and decreased efficiency in palmitate-perfused hyperthyroid rat heart.</a> AJP - Heart. 280, 977-983 (2001).</li><li>Fontaine, E., Eriksson, O., Ichas, F., Bernardi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+Permeability+Transition+Pore+in+Skeletal+Muscle+Mitochondria.">Regulation of the Permeability Transition Pore in Skeletal Muscle Mitochondria.</a> J. Biol. Chem. 273, 12662-12668 (1998).</li><li>Berman, S. B., Watkins, S. C., Hastings, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+biochemical+and+ultrastructural+comparison+of+mitochondrial+permeability+transition+in+isolated+brain+and+liver+mitochondria:+evidence+for+reduced+sensitivity+of+brain+mitochondria.">Quantitative biochemical and ultrastructural comparison of mitochondrial permeability transition in isolated brain and liver mitochondria: evidence for reduced sensitivity of brain mitochondria.</a> Exp. Neurol. 164, 415-425 (2000).</li><li>Panov, A., Dikalov, S., Shalbuyeva, N., Hemendinger, R., Greenamyre, J. T., Rosenfeld, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species-+and+tissue-specific+relationships+between+mitochondrial+permeability+transition+and+generation+of+ROS+in+brain+and+liver+mitochondria+of+rats+and+mice.">Species- and tissue-specific relationships between mitochondrial permeability transition and generation of ROS in brain and liver mitochondria of rats and mice.</a> Am. J. Physiol. Cell Physiol. 292, 708-718 (2007).</li><li>Frezza, C., Cipolat, S., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+isolation:+functional+mitochondria+from+mouse+liver,+muscle+and+cultured+filroblasts.">Organelle isolation: functional mitochondria from mouse liver, muscle and cultured filroblasts.</a> Nature Protocols. 2, 287-295 (2007).</li><li>Pallotti, F., Lenaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subfractionation+of+mitochondria+from+animal+cells+and+tissue+culture+lines.">Isolation and subfractionation of mitochondria from animal cells and tissue culture lines.</a> Methods Cell Biol. 80, 3-44 (2007).</li></ol>

אין ניגודי האינטרסים הכריזו.

הפרוטוקול המובא כאן מתאר את הצעדים הניסיוניים הדרושים כדי להעריך פתיחה נקבובית מעבר חדירות בלב מיטוכונדריה מבודד (איור 1 ואיור 4): ההליך לבידוד לב העכבר מיטוכונדריה, פקדי נשימה המבטיחים יושרה והפונקציונלית שלהם, פרמטרים המיטוכונדרי במעקב במהלך פתיחת mtPTP ואת הצבעים המועסקים למדידתם, הקמת מכשור spectrofluorometric, והאפיון של פתיחת mtPTP. Spectrofluorometer המועסק בפרוטוקול זה מאפשר מדידה מהירה של השלושה הפרמטרים הללו בו זמנית. עם זאת, הפרוטוקול הבסיסי יכול להיות מועסק באמצעות מכשירים אחרים זמינים באופן מסחרי, אם כי ברזולוצית זמן ירד. צבעים ללא ratiometric יכולים גם להיות מועסקים, אך אינו מתירים מדדי כמותיים של שניהם Ca 2 + ופוטנציאל הקרום המיטוכונדריה 5. הכנת המיטוכונדריה איכות טובה היא חיונית כאשר מנסים לאמוד את פתיחת mtPTP, שכן הן היכולת לצבור Ca 2 + ולשמור על פוטנציאל הממברנה הוא חוזקה גומלין. לדוגמה, מצא כי המיטוכונדריה עם תערוכת RCR נמוכה המופחתת הצטברות JC-1 והעניים Ca 2 + גיוס. ההליך אנו משתמשים לבודד לב המיטוכונדריה הותאם מ 6 פרוטוקולים שפורסמו בעבר, 7 ו הוכיח להיות מאוד עקבי ביצירה באיכות גבוהה, כפי שנמדד על ידי המיטוכונדריה בדיקות ציטוכרום c RCR ו. פתיחת mtPTP היא תופעה מורכבת שכרוכות בשינויים בכמה רמות באנטומיה והפיסיולוגיה של המיטוכונדריה. Assay מתואר כאן spectrofluorometrically מודד את השינויים החלים בו זמנית בextramitochondrial Ca 2 + רמות, קרום הפוטנציאל המיטוכונדריה והנפח של המיטוכונדריה. המדידה של מספר פרמטרים באותו הזמן מציעה יותר compreheתמונת nsive של התופעה ממדידת פרמטרים בודדים בלבד, כגון Ca 2 + גיוס או נפיחות. יתר על כן, השימוש בצבעי ratiometric Fura FF וJC-1 לCa 2 + ומדידות קרום פוטנציאליות מגביל חפץ הקשור לניסוי לצבוע את הריכוז, ספיג או photobleaching. צבעים אלה יכולים גם להיות מכוילים בקלות כדי להשיג מדידות כמותיות יותר. תצפית חשובה היא שכל ריכוזי הצבע צריך להישמר נמוך ככל האפשר, כדי למנוע הפרעה בנשימת המיטוכונדריה ופוטנציאל קרום כמו גם לרגש פתיחת mtPTP. בידות שלנו, ריכוזים JC-1 מעל 1 מיקרומטר לצמצם את מספר הפעימות הדרושות כדי לפתוח mtPTP ידי 1-2 פעימות. פקדים לכן מתאימים צריכים להתבצע על ידי נטרול הצבעים והקלטת פרמטרים אחרים (כגון נפיחות לבד) כדי להיות בטוח שמספר הפעימות הדרושות כדי לפתוח את PTP נשאר עקבי. לאורך פרוטוקול זהיש לנו נמדדנו פתיחת mtPTP למיטוכונדריה הזעה בsuccinate המצע השני המורכב בנוכחות rotenone, אשר מעכב העברת אלקטרונים הפוכה מהשני מורכבת למצעים אחרים בדרכי נשימה מורכבים I. יכול לשמש היטב, כגון פירובט / malate התומך בהנשמה מורכבת י עם זאת, הבדלי מצע הקשורים במספר של Ca 2 + פולסים נדרשים לפתוח mtPTP דווח 8. באותו הפרוטוקול למדידת פתיחת mtPTP יכול להיות מועסק במיטוכונדריה מבודדת מרקמות שונות או קווים סלולריים. יש לנו מנוצלים בהצלחה בפרוטוקול זה לכבד, בשרירים ובמוח מיטוכונדריה, למרות שהמספר של Ca 2 + פולסים הדרושים כדי לעורר את פתיחת mtPTP הייתה רקמה תלויה כפי שדווח 9, 10 בעבר. פרוטוקול בידוד המיטוכונדריה חייב להיות מותאם ומותאם לכל שורת רקמה או תא עקב דרישות ספציפיות בנוגע להליך הומוגניזציה וחוצצי בידוד 11, 12 </sup&gt;.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם המגיב </td><td> חברה </td><td> מספר קטלוגים </td></tr><tr><td> טריפסין </td><td> סיגמה אולדריץ </td><td> T3030 </td></tr><tr><td> טריפסין המעכב (סויה) </td><td> סיגמה אולדריץ </td><td> T9128 </td></tr><tr><td> hydrosulfite נתרן </td><td> סיגמה אולדריץ </td><td> 71699 </td></tr><tr><td> Rotenone </td><td> סיגמה אולדריץ </td><td> R8875 </td></tr><tr><td> ציטוכרום C </td><td> סיגמה אולדריץ </td><td> C7752 </td></tr><tr><td> Alamethicin </td><td> סיגמה אולדריץ </td><td> A4665 </td></tr><tr><td> CCCP </td><td> סיגמה אולדריץ </td><td> C2759 </td></tr><tr><td> ציקלוספורין </td><td> Calbiochem </td><td> 239835 </td></tr><tr><td> Fura FF </td><td> Invitrogen </td><td> F14180 </td></tr><tr> &lt;td&gt; JC-1 </td><td> Invitrogen </td><td> T3168 </td></tr><tr><td> מטחנת רקמה פוטר-Elvehjem עם טפלון העלי 15 מ&quot;ל </td><td> תעשיות ויטון </td><td></td></tr><tr><td> stirrer העילי </td><td> תעשיות ויטון </td><td> 903475 </td></tr><tr><td> Oxytherm (אלקטרודה חמצן הטמפרטורה המבוקרת) </td><td> Hansatech מכשירים </td><td></td></tr><tr><td> spectrofluorometer הפליטה QuantaMaster 80 כפול </td><td> פוטון טכנולוגית International, Inc </td><td></td></tr></tbody></table>

multi-parameter measurement of the permeability transition pore opening in isolated mouse heart mitochondria

נקבובי מעבר חדירות המיטוכונדריה (mtPTP) הוא ערוץ מסוים שאינם נוצר בקרום המיטוכונדריה הפנימי להובלה מומסת עם מסה מולקולרית קטנה מ -1.5 kDa. למרות הזהות המוחלטת המולקולרית של נקבובי היא עדיין תחת דיון, חלבונים כגון cyclophilin D, VDAC וANT לתרום להיווצרות mtPTP. למרות המעורבות של פתיחת mtPTP במוות של תאים מבוססת היטב 1, מצטבר ראיות מצביע על כך שmtPTP משרת תפקיד פיסיולוגי במהלך המיטוכונדריה Ca 2 + הומאוסטזיס 2, bioenergetics וחמזור איתות 3. פתיחת mtPTP מופעלת על ידי מטריקס Ca 2 + אבל פעילותו יכולה להיות מווסתת על ידי מספר גורמים אחרים כגון סטרס חמצונים, דלדול נוקלאוטיד אדנין, ריכוזים גבוהים של פי, שלילת קוטביות הקרום המיטוכונדריה או תרה, וחומצות שומן ארוכת שרשרת 4. במבחנה, mtPTP פתיחה יכולה להיות achieved על ידי הגדלת ריכוז + Ca 2 בתוך מטריצת המיטוכונדריה באמצעות תוספת חיצונית של Ca 2 + (קיבולת אצירת סידן). כאשר Ca 2 + רמות בתוך המיטוכונדריה מגיע לסף מסוים, ומאפשר mtPTP פותח Ca 2 + שחרור, פיזורו של כוח מניע הפרוטונים, קריסת פוטנציאל קרום וגידול בהיקף מטריקס המיטוכונדריה (נפיחות) שסופו של דבר מוביל לקרע של קרום חיצוני של המיטוכונדריה ואובדן בלתי הפיך של תפקוד אברון. כאן אנו מתארים assay fluorometric המאפשר אפיון מקיף של פתיחת mtPTP בלב עכבר מבודד המיטוכונדריה. הבדיקה כרוכה במדידה בו הזמנית של 3 פרמטרי המיטוכונדריה ששינו כאשר פתיחת mtPTP מתרחשת: המיטוכונדריה Ca 2 + הטיפול (ספיגה ושחרורו, כפי שהיא נמדדת על ידי Ca 2 + ריכוז במדיום assay) פוטנציאלית, מיטוכונדריה ממברנה, וmitochoנפח ndrial. את הצבעים המועסקים על Ca 2 + מדידה במדיום assay ופוטנציאל הקרום המיטוכונדריה הם Fura FF, impermeant קרום, מחוון ratiometric שעובר שינוי באורך גל העירור בנוכחות Ca 2 +, וJC-1, קטיוני, מחוון ratiometric המהווה מונומרים ירוקים או אדומים אגרגטים בפוטנציאל קרום נמוך וגבוה, בהתאמה. שינויים בהיקף המיטוכונדריה נמדדים על ידי הקלטת פיזור אור על ידי ההשעיה של המיטוכונדריה. מאז באיכות גבוהה, המיטוכונדריה פונקציונלית נדרשות לבדיקת פתיחת mtPTP, אנחנו גם מתארים את הצעדים הדרושים כדי להשיג לב שלם, בשילוב ומאוד פונקציונלי מבודד המיטוכונדריה.

Watch this Scientific Journal Video about Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria at JoVE.com

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

פרוטוקול spectrofluorometric למדידת פתיחת מעבר המיטוכונדריה חדירות הנקבובית בלב עכבר מבודד המיטוכונדריה מוצג כאן. הבדיקה כרוכה במדידה בו הזמנית של Ca מיטוכונדריה<sup&gt; 2 +</sup&gt; טיפול, קרום נפח פוטנציאלי ומיטוכונדריה המיטוכונדריה. ההליך לקבלת לב איכותי ופונקציונלי המיטוכונדריה הוא גם תאר.

מדידת פרמטר-Multi של הפתיחה הנקבובית מעבר החדירות בלב עכבר מבודד המיטוכונדריה

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

multi-parameter-measurement-permeability-transition-pore-opening

Google

Research

JoVE Journal

Biology

21.4K Views.  University of Washington, Seattle. פרוטוקול spectrofluorometric למדידת פתיחת מעבר המיטוכונדריה חדירות הנקבובית בלב עכבר מבודד המיטוכונדריה מוצג כאן. הבדיקה כרוכה במדידה בו הזמנית של Ca מיטוכונדריה 2 + טיפול, קרום נפח פוטנציאלי ומיטוכונדריה המיטוכונדריה. ההליך לקבלת לב איכותי ופונקציונלי המיטוכו...

Video: Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

הכנה של הלב עכברוש מצמידים מאוד המיטוכונדריה

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

ההדמיה confocal של הבזקים בודדים מיטוכונדריאלי Superoxide בלב שלם או<em&gt; בVivo</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

כימות יחיד כלי דם קטנים בחדירות מבודדת-perfused דם עכברוש ריאות הכנה

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

מדידת אוסמוטי המים החדירות מקדם (P<sub&gt; F</sub&gt;) של תאים כדוריים: Protoplasts צמח מבודד כדוגמה

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

Immunodetection של החלבונים בממברנה חיצוניים על ידי cytometry הזרימה של מיטוכונדריה מבודדת

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

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.  ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

בידוד של המיטוכונדריה מכמויות מינימליות של שרירי שלד עכבר למדידות תפוקה גבוהה microplate נשימה

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

שיטות לקביעת המחירים של גלוקוז ו חומצות שומן חמצון שבלב עכברוש העבודה המבודד

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

טכניקת כלית עכבר המבודדת perfused

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found responsible for very diverse diseases, ranging from neurological pathologies to cancer. Interestingly, mitochondria have recently been shown to display the capacity to be transferred between cell types, notably from human mesenchymal stem cells (MSC) to cancer cells in coculture conditions, with metabolic and functional consequences for the mitochondria recipient cells, further enhancing the current interest for the biological properties of these organelles.
Evaluating the effects of the transferred MSC mitochondria in the target cells is of primary importance to understand the biological outcome of such cell-cell interactions. The MitoCeption protocol described here allows the transfer of the mitochondria isolated beforehand from the donor cells to the target cells, using MSC mitochondria and glioblastoma stem cells (GSC) as a model system. This protocol has previously been used to transfer mitochondria, isolated from MSCs, to adherent MDA-MB-231 cancer cells. This mitochondria transfer protocol is adapted here for GSCs that present the specific particularity of growing as neurospheres in vitro. The transfer of the isolated mitochondria can be followed by fluorescence-activated cell sorting (FACS) and confocal imaging using mitochondria vital dyes. The use of mitochondria donor and target cells with distinct haplotypes (SNPs) also allows detection of the transferred mitochondria based on the concentration of their circular mitochondrial DNA (mtDNA) in the target cells. Once the protocol has been validated with these criteria, the cells harboring the transferred mitochondria can be further analyzed to determine the effects of the exogenous mitochondria on biological properties such as cell metabolism, plasticity, proliferation and response to therapy.

Here, a protocol (MitoCeption) is presented to transfer mitochondria, isolated from human mesenchymal stem cells (MSC), to glioblastoma stem cells (GSC), with the goal of studying their biological effects on GSC metabolism and functions. A similar protocol can be adapted to transfer mitochondria between other cell types.