מחקר זה מומן על ידי מכוני הבריאות הלאומיים מענק R01HL086939 (NCC).

<ol>
	<li>Pace, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sympathetic+control+of+pulmonary+vascular+impedance+in+anesthetized+dogs.">Sympathetic control of pulmonary vascular impedance in anesthetized dogs.</a> Circ Res. 29, 555-568 (1971).</li><li>Ewalenko, P., Stefanidis, C., Holoye, A., Brimioulle, S., Naeije, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+vascular+impedance+vs.+resistance+in+hypoxic+and+hyperoxic+dogs:+effects+of+propofol+and+isoflurane.">Pulmonary vascular impedance vs. resistance in hypoxic and hyperoxic dogs: effects of propofol and isoflurane.</a> J Appl Physiol. 74, 2188-2193 (1993).</li><li>Nichols, W. W., O'Rourke, M. F., Hartley, C., McDonald, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> McDonald's blood flow in arteries : theoretical, experimental, and clinical principles. , (2005).</li><li>Tuchscherer, H. A., Webster, E. B., Chesler, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+Vascular+Resistance+and+Impedance+in+Isolated+Mouse+Lungs:+Effects+of+Pulmonary+Emboli.">Pulmonary Vascular Resistance and Impedance in Isolated Mouse Lungs: Effects of Pulmonary Emboli.</a> Annals of Biomedical Engineering. 34, 660-668 (2006).</li><li>Tuchscherer, H. A., Vanderpool, R. R., Chesler, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+vascular+remodeling+in+isolated+mouse+lungs:+Effects+on+pulsatile+pressure-flow+relationships.">Pulmonary vascular remodeling in isolated mouse lungs: Effects on pulsatile pressure-flow relationships.</a> Journal of Biomechanics. 40, 993-1001 (2007).</li><li>Vanderpool, R., Naeije, R., Chesler, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impedance+in+Isolated+Mouse+Lungs+for+the+Determination+of+Site+of+Action+of+Vasoactive+Agents+and+Disease.">Impedance in Isolated Mouse Lungs for the Determination of Site of Action of Vasoactive Agents and Disease.</a> Ann Biomed Eng. , (2010).</li><li>Vanderpool, R., Kim, A., Molthen, R., Chesler, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+acute+rho+kinase+inhibition+on+chronic+hypoxia-induced+changes+in+proximal+and+distal+pulmonary+arterial+structure+and+function.">Effects of acute rho kinase inhibition on chronic hypoxia-induced changes in proximal and distal pulmonary arterial structure and function.</a> Journal of Applied Physiology. , (2010).</li><li>El-Bizri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smooth+muscle+protein+22alpha-mediated+patchy+deletion+of+Bmpr1a+impairs+cardiac+contractility+but+protects+against+pulmonary+vascular+remodeling.">Smooth muscle protein 22alpha-mediated patchy deletion of Bmpr1a impairs cardiac contractility but protects against pulmonary vascular remodeling.</a> Circ Res. 102, 380-388 (2008).</li><li>Champion, H., Michelakis, E., Hassoun, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+invasive+and+noninvasive+approach+to+the+right+ventricle-pulmonary+circulation+unit:+state+of+the+art+and+clinical+and+research+implications.">Comprehensive invasive and noninvasive approach to the right ventricle-pulmonary circulation unit: state of the art and clinical and research implications.</a> Circulation. 120, 992-1007 (2009).</li><li>Gan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasively+assessed+pulmonary+artery+stiffness+predicts+mortality+in+pulmonary+arterial+hypertension.">Noninvasively assessed pulmonary artery stiffness predicts mortality in pulmonary arterial hypertension.</a> Chest. 132, 1906-1912 (2007).</li><li>Mahapatra, S., Nishimura, R., Sorajja, P., Cha, S., McGoon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+pulmonary+arterial+capacitance+and+mortality+in+idiopathic+pulmonary+arterial+hypertension.">Relationship of pulmonary arterial capacitance and mortality in idiopathic pulmonary arterial hypertension.</a> J Am Coll Cardiol. 47, 799-803 (2006).</li><li>Bogaard, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+pulmonary+artery+pressure+elevation+is+insufficient+to+explain+right+heart+failure.">Chronic pulmonary artery pressure elevation is insufficient to explain right heart failure.</a> Circulation. 120, 1951-1960 (2009).</li></ol>

אין ניגודי אינטרסים הכריז.

צעדים קריטי הניתוח זה קריטי, כי הטיפול נלקח כאשר חותך את הצלעות מן הריאות. הריאות חייב להיות חשוף באופן מלא מעצורים על ידי הרקמות הסובבות במהלך האינפלציה, אך לא נפגע במהלך תהליך של בידוד. השימוש של אובייקט שטוח כמו בקצה האחורי של מל?...

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<td>1 ml syringe</td>
<td>&nbsp;</td>
<td>Fisher Sci</td>
<td>14-829-10F</td>
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<td>10 ml syringe</td>
<td>&nbsp;</td>
<td>Fisher Sci</td>
<td>14-823-2A </td>
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<td>60 ml syringe</td>
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<td>Fisher Sci</td>
<td>13-689-8 </td>
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<td>RPMI with GLN 6/PK</td>
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<td>MT10040CV </td>
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<td>IL-1 Isolated perfused mouse lung system </td>
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<td>Blood Pressure Transducer P75 for PLUGSYS Module</td>
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<td>ME 4 PXN Precision PXN Inline Flowsensors</td>
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<td>Cole-Parmer Multi-Syringe Pumps</td>
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<td>Cole-Parmer </td>
<td>EW-74900-20</td>
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<td>Nembutal 50MG/ML 20ML Vial</td>
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<td>Amatheon</td>
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characterization of the isolated, ventilated, and instrumented mouse lung perfused with pulsatile flow

מבודד, מאוורר instrumented הכנה עכבר הריאות מאפשר יציב pulsatile כלי דם ריאתי לחץ זרימה יחסים כדי להימדד עם שליטה עצמאית על שיעור ריאתי עורקי זרימה, קצב זרימת waveform, לחץ האוויר ואת הלחץ פרוזדורים שמאל. התנגדות כלי דם ריאתי מחושב על בסיס ריבוי נקודות, לחץ זרימת עקומות יציב; עכבה כלי דם ריאתי מחושב מלחץ זרימת עקומות pulsatile להשיג בחנות מגוון רחב של תדרים. כמו מוכר עכשיו קלינית, עכבה היא מדד מעולה של afterload חדרית הזכות מאשר התנגדות משום שהיא כוללת את ההשפעות של ציות כלי הדם, אשר אינן זניחות, במיוחד במחזור ריאתי. שלושה מדדים חשובים של עכבה - אפס הרץ עכבה Z 0, העכבה האופיינית Z C, מדד גל השתקפות R W - לספק תובנה באזור דיסטלי חתך עורקי עבור זרימה, קשיות העורקים הפרוקסימלי ואת עכבה במעלה הזרם, במורד הזרם אי התאמה, בהתאמה. כל התוצאות שהתקבלו מבודד, מאוורר והריאות perfused שאינן תלויות הטון האוהד של מערכת העצבים, מצב עוצמת ההשפעה של הרדמה. השתמשנו בטכניקה זו כדי לכמת את ההשפעה של תסחיפים ריאתי היפוקסיה כרונית על התנגדות עכבה, וכדי להבדיל בין אתרי פעולה (כלומר, לעומת הפרוקסימלית הדיסטלית) של סוכנים vasoactive ומחלות באמצעות תלות בלחץ של Z-C. יתר על כן, כאשר טכניקות אלה משמשים עם הריאות של זנים מהונדסים גנטית של עכברים, תופעות של ליקויים ברמה המולקולרית על מבנה כלי הדם ואת תפקוד ריאתי ניתן לקבוע.

Watch this Scientific Journal Video about Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow at JoVE.com

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

פרוטוקול הבא מתאר את התהליך של בידוד, איוורור ו instrumenting הריאות העכבר כדי למדוד יציב או pulsatile לחץ הדם הריאתי זרימה יחסים כדי לכמת את ההשפעות של זרימת הדם, זרימת אוויר, שינויים בדרכי הנשימה ושינויים בכלי הדם על afterload חדרית הנכון.

אפיון מבודד, מאוורר ריאות, instrumented ועכבר perfused עם תזרים pulsatile

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

characterization-isolated-ventilated-instrumented-mouse-lung-perfused

Research

JoVE Journal

Biology

16.2K Views.  University of Wisconsin – Madison. פרוטוקול הבא מתאר את התהליך של בידוד, איוורור ו instrumenting הריאות העכבר כדי למדוד יציב או pulsatile לחץ הדם הריאתי זרימה יחסים כדי לכמת את ההשפעות של זרימת הדם, זרימת אוויר, שינויים בדרכי הנשימה ושינויים בכלי הדם על afterload חדרית הנכון.

Video: Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Cytometry זרימה טיהור תאים עכבר meiotic

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

מיקרו מכונות אפיון רקמת הריאה באמצעות מיקרוסקופ כוח אטומי

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation &amp; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

בידוד &amp; אפיון Hoechst נמוך CD45 שלילי עכבר mesenchymal ריאות בתאי גזע

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

דור של עכברים שמקורם בתאי גזע pluripotent מושרים

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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 ...

An Immunofluorescent Method for Characterization of Barrett&#8217;s Esophagus Cells

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One of the primary risk factors of EAC is Barrett&#8217;s esophagus (BE), a metaplastic change of the normal squamous esophagus in response to chronic heartburn. Despite the well-established connection between EAC and BE, interrogation of the molecular events, particularly altered signaling pathways involving progression of BE to EAC, are poorly understood. Much of this is due to the lack of suitable in vitro models available to study these diseases. Recently, immortalized BE cell lines have become commercially available allowing for in vitro studies of BE. Here, we present a method for immunofluorescent staining of immortalized BE cell lines, allowing in vitro characterization of cell signaling and structure after exposure to therapeutic compounds. Application of these techniques will help develop insight into the mechanisms involved in BE to EAC progression and provide potential avenues for treatment and prevention of EAC.

An Immunofluorescent Method for Characterization of Barrett&amp;#8217;s Esophagus Cells

There is a discernible need for improved information on molecular drivers of Barrett&#8217;s Esophagus. Immunofluorescent staining is a useful technique for understanding the effects of cell signaling on cell morphology. We present a simple, effective protocol for the use of immunofluorescent staining to assess therapeutic treatment in Barrett&#8217;s Esophagus cells.

שיטת Immunofluorescent לאפיון של תאי הוושט של בארט

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One ...

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods are needed that can quantify the pathologic changes. Furthermore, to provide translational relevance to the mouse models, such measurements should be tests that can easily be done in both humans and mice. Unfortunately, in the present literature few phenotypic measurements of lung function have direct application to humans. One exception is the diffusing capacity for carbon monoxide, which is a measurement that is routinely done in humans. In the present report, we describe a means to quickly and simply measure this diffusing capacity in mice. The procedure involves brief lung inflation with tracer gases in an anesthetized mouse, followed by a 1 min gas analysis time. We have tested the ability of this method to detect several lung pathologies, including emphysema, fibrosis, acute lung injury, and influenza and fungal lung infections, as well as monitoring lung maturation in young pups. Results show significant decreases in all the lung pathologies, as well as an increase in the diffusing capacity with lung maturation. This measurement of lung diffusing capacity thus provides a pulmonary function test that has broad application with its ability to detect phenotypic structural changes with most of the existing pathologic lung models.

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

פונקצית phenotyping עכבר ריאתי<em&gt; In vivo</em&gt; עם קיבולת הריאות לשדר

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods ...

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 ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

מדידת Endoreduplication על ידי Cytometry זרימה של פקעת מבודד Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.