Diese Experimente wurden von einem Labor Handbuch, das in einem Kurs verwendet wurde, orchestriert von Dr. HL Atwood, am Institut für Zoologie, Universität Toronto geändert. Die Übungen wurden auch genutzt und modifiziert aus einem Handbuch, das für &quot;6 th INTENSIVE IBRO WORKSHOP ON BASIC Neuroscience&quot; produziert wurde und an der Korea University, Seoul, South Korea im Jahr 1993 (Cooper et al., 1993) statt. Die aktuellen Änderungen waren erforderlich, um Geräte gemeinsam nutzen zu Tag Schüler gerichtet Labors an verschiedenen Universitäten präsentieren. Unterstützt von der University of Kentucky, Department of Biology, Office of Undergraduate Studies and College of Arts &amp; Sciences.

<ol>
	<li>Bennett, M. V. L., Barrio, L. C., Bargiello, T. A., Spray, D. C., Hertzberg, E., Sdez, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctions:+new+tools,+new+answers,+new+questions.">Gap junctions: new tools, new answers, new questions.</a> Neuron. 6, 305-320 (1991).</li><li>Bernardini, G., Peracchia, C., Peracchia, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+effects+of+heptanol+on+gap+junction+structure+and+cell-to-cell+electrical+coupling.">Reversible effects of heptanol on gap junction structure and cell-to-cell electrical coupling.</a> European Journal of Cell Biology. 34 (2), 307-312 (1984).</li><li>Cooper, R. L., Chang, J. J., Ito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+report+on+the,+"SIXTH+INTENSIVE+IBRO+WORKSHOP+ON+BASIC+NEUROSCIENCE".">A report on the, "SIXTH INTENSIVE IBRO WORKSHOP ON BASIC NEUROSCIENCE".</a> , 116-116 (1985).</li><li>Cragg, B. G., Thomas, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+conduction+velocity+and+the+diameter+and+internodal+length+of+peripheral+nerve+fibers..">The relationship between conduction velocity and the diameter and internodal length of peripheral nerve fibers..</a> Journal of Physiology. 136, 606-614 (1957).</li><li>Erlanger, J. G. a. s. s. e. r., S, H., Bishop, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+compound+nature+of+the+action+current+of+nerves+as+disclosed+by+the+cathode+ray+oscillograph.">The compound nature of the action current of nerves as disclosed by the cathode ray oscillograph.</a> American Journal of Physiology. 70, 624-666 (1924).</li><li>Furshpan, E. J., Potter, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+at+the+giant+motor+synapses+of+the+crayfish.">Transmission at the giant motor synapses of the crayfish.</a> Journal of Physiology. 145 (2), 289-325 (1959).</li><li>Johnston, M. F., Simon, S. A., Ramrn, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+anesthetics+with+electrical+synapses.">Interaction of anesthetics with electrical synapses.</a> Nature (Lond). 286, 498-500 (1980).</li><li>Loewenstein, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+of+membrane+junctions.">Permeability of membrane junctions.</a> Annual NY Academy of Sciences. 137, 441-472 (1966).</li><li>Meda, P., Bruzzone, R., Knodel, S., Orci, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockage+of+cell-to-cell+communication+within+pancreatic+acini+is+associated+with+increased+basal+release+of+amylase.">Blockage of cell-to-cell communication within pancreatic acini is associated with increased basal release of amylase.</a> Journal of Cell Biology. 103 (2), 475-483 (1986).</li><li>Peracchia, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+gap+junction+resistance+with+acidification+in+crayfish+septate+axons+is+closely+related+to+changes+in+intracellular+calcium+but+not+hydrogen+ion+concentration.">Increase in gap junction resistance with acidification in crayfish septate axons is closely related to changes in intracellular calcium but not hydrogen ion concentration.</a> Journal of Membrane Biology. 113 (1), 75-92 (1990).</li><li>Peracchia, C., Dulhunty, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+resistance+junctions+in+crayfish:+structural+changes+with+functional+uncoupling.">Low resistance junctions in crayfish: structural changes with functional uncoupling.</a> Journal of Cell Biology. 70, 419-439 (1976).</li><li>Peracchia, C., Bernardini, G., Peracchia, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+calmodulin+involved+in+the+regulation+of+gap+junction+permeability.">Is calmodulin involved in the regulation of gap junction permeability.</a> Pf&#252;gers Arch. 399, 152-154 (1983).</li><li>Peracchia, C., Lazrak, A., Peracchia, L. L., Peracchia, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+models+of+channel+interaction+and+gating+in+gap+junctions.">Molecular models of channel interaction and gating in gap junctions.</a> Handbook of Membrane Channels. Molecular and Cellular Physiology. , 361-377 (1994).</li><li>Spray, D. C., Harris, A. L., Bennett, M. V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctional+conductance+is+a+simple+and+sensitive+function+of+intracellular+pH.">Gap junctional conductance is a simple and sensitive function of intracellular pH.</a> Sciences NY. 211, 712-715 (1981).</li><li>Spray, D. C., Harris, L. L., Bennett, M. V. L., Nuccitelli, R., Deamer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+pH+and+Ca+dependence+of+gap+junctional+conductance.">Comparison of pH and Ca dependence of gap junctional conductance.</a> Intracellular pH: Its Measurement, Regulation, and Utilization in Cellular Functions. , 445-461 (1982).</li><li>Spray, D. C., White, R., De Carvalho, C., Harris, A. L., Bennett, M. L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gating+of+gap+junction+channels.">Gating of gap junction channels.</a> Journal of Biophysics. 45, 219-230 (1984).</li><li>Watanabe, A., Grundfest, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impulse+propagation+at+the+septal+and+commissural+junctions+of+crayfish+lateral+giant+axons.">Impulse propagation at the septal and commissural junctions of crayfish lateral giant axons.</a> Journal of General Physiology. 45, 267-308 (1961).</li><li>Wiersma, C. A. G., Hughes, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+functional+anatomy+of+neuronal+units+in+the+abdominal+cord+of+the+crayfish,+Procambarus+clarkii.">On the functional anatomy of neuronal units in the abdominal cord of the crayfish, Procambarus clarkii.</a> Journal of Comparative Neurology. 116, 209-228 (1961).</li></ol>

Unser Ziel in der Online-Video-Präsentation und das Papier ist zu zeigen, dass die biophysikalischen Eigenschaften von Zellen, können zum Teil als elektrische Schaltungen modelliert werden. Darüber hinaus, mit Live-Nervengewebe, die relativ leicht zu erhalten ist, sind grundlegende Prinzipien der Leitgeschwindigkeit Refraktärzeiten und elektrophysiologische Aufnahmetechniken für Bachelor-Student Labors mit bescheidenen Investitionen von Geräten möglich. Die Themen und grundlegende Paradigmen vorgestellt leicht für die Anforderungen der verschiedenen Kurse geändert werden. Wartung von Flusskrebsen und ihren Reichtum macht sie attraktiver Modelle für Schüler bestimmte Experimente. Krustentier ventrale Nervenstränge sind in der Regel robust und behalten physiologische Integrität in einer minimalen Kochsalzlösung für Stunden, was ausreichend für eine 3-stündige Schülerlabor ist. Da einige der großen Axone in den VNC des Krebses über Gap Junctions, zusätzliche Experimente auf ihren Beitrag durchgeführt werden können, und andere Eigenschaften als gefunden in der Standard-Frosch Ischiasnerv Vorbereitung nachgewiesen werden können, sind verbunden. Der Ischiasnerv ist ein klassisches Modell für die Bewältigung Verbindung Aktionspotentiale und Leitungseigenschaften. Es könnte sogar eine interessante Vergleichsversuch für Studenten Leitungseigenschaften vergleichen, Axon Rekrutierung und Refraktärzeiten zwischen diesen beiden Präparaten werden.

Leiterplatte <ol><li> Electronics unten für die breadboard Experimente aufgeführt sind, können bei einem lokalen Elektronik-Geschäft wie Radio Shack erhältlich. </li><li> Bread Board, Widerstände, Kondensatoren, Batterien und Drähte, die als Knotenpunkte für das Steckbrett dienen kann, wie in den Experimenten dargestellt. </li><li> Eine gemeinsame Voltmeter (Wavetek Meterman Voltmeter) </li><li> A / D-Karte für Online-Aufzeichnung auf einem Computer. Elektrische Signale werden auf Linie zu einem PowerLab/4s Schnittstelle (ADInstruments, Colorado Springs, CO, USA) aufgezeichnet. Wir verwenden Standard-Software von ADInstruments namens Chart-oder Scope. </li></ol> Physiologie Experimente <ol><li> Crayfish (Procambarus clarkii). Atchafalaya Biological Supply Co., Raceland, LA., USA. </li><li> Standard-Krebse Kochsalzlösung: Geändert von Van Harreveld-Lösung (1936). (In mm) 205 NaCl, 5,3 KCl, 13,5 CaCl 2 · 2H 2 O, 2,45 MgCl 2 6H 2 O, 5 HEPES und auf pH 7,4 eingestellt. Alle Kochsalzlösung Chemikalien wurden von Sigma Chemical Company (St. Louis, MO) erhalten. </li><li> Dissection-Tools: Fine Nr. 5 Pinzette, feine Schere, Messer Messerhalter, # 26002-20 Insektennadeln (alle von Fine Science Tools (USA), Inc., 373-G Vintage Park Drive, Foster City, CA 94404-1139 erhalten) </li><li> Ein Nerv Kammer Schale (ADInstruments, Colorado Springs, CO, USA) auf die Nerven zu behalten. Die Drähte, die mit den Nerven Kammer Gericht kommen kann verwendet werden, um die Nerven zu stimulieren. </li><li> Ein Saugelektrode wird verwendet, um die Signale aufzuzeichnen. </li><li> Der Manipulator oder einer Klammer an einem Ring stehen auch als Halterung für die Saug-Elektrode dienen. </li><li> Faraday-Käfig </li><li> Präpariermikroskop </li><li> High Intensity Illuminator (Lichtquelle) </li><li> Mikroskop-Plattform </li><li> AC / DC Differenzverstärker (AM Systems Inc. Modell 3000) </li><li> PowerLab 26T (AD Instruments) </li><li> Extrazelluläre Verstärker (AD Instruments) </li><li> LabChart 7 (ADI Instruments) </li><li> Dissecting Werkzeuge </li><li> Ein Ring stehen und Klemme als Halterung für die Aufnahme Saugelektrode dienen </li></ol>

modeling biological membranes with circuit boards and measuring electrical signals in axons: student laboratory exercises

Dies ist eine Demonstration, wie elektrische Modelle können verwendet werden, um biologische Membranen zu charakterisieren. Diese Übung führt auch biophysikalische Terminologie in Elektrophysiologie. Das gleiche Gerät ist in der Membran-Modell als zu leben Zubereitungen verwendet. Einige Eigenschaften eines isolierten Nervenstrang untersucht: Nervenaktionspotentiale, Rekrutierung von Neuronen und Reaktionsfähigkeit der Nervenstrang auf Umweltfaktoren.

Watch this Scientific Journal Video about Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises at JoVE.com

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

Dies ist ein Beispiel dafür, wie biologische Membranen verstanden mit elektrischen Modelle. Wir zeigen auch Verfahren zur Erfassung Aktionspotentiale aus dem Bauchmark der Krebse für Schüler orientiert Labors.

Modellierung biologischer Membranen mit Leiterplatten und Messung elektrischer Signale in Axone: Schülerlabor Übungen

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

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22.4K Aufrufe.  University of Kentucky. Dies ist ein Beispiel dafür, wie biologische Membranen verstanden mit elektrischen Modelle. Wir zeigen auch Verfahren zur Erfassung Aktionspotentiale aus dem Bauchmark der Krebse für Schüler orientiert Labors.

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Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by &Delta;&Phi;, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of &Delta;&Phi; on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of &Delta;&Phi;. This video shows the protocol for measuring &Delta;&Phi; using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of &Delta;&Phi;.

External electric field induces a voltage on the membrane of a cell, termed the induced membrane voltage (&Delta;&Phi;). By using the potentiometric dye di-8-ANEPPS, it is possible to measure the &Delta;&Phi; noninvasively. This video shows the protocol for measuring &Delta;&Phi; using di-8-ANEPPS.

Messung der induzierten Spannung Membran mit Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell ...

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

Computer-assisted Großflächige Visualisierung und Quantifizierung der Inselzellen der Bauchspeicheldrüse Masse, Größenverteilung und Architektur

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

High-Throughput-Screening und Biosensorik mit Fluorescent C. elegans Die Stämme

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

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.

Mess-Fast-Flux Calcium in Kardiomyozyten

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

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Mess räumliche und zeitliche Ca<sup&gt; 2 +</sup&gt; Signale in Arabidopsis-Pflanzen

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments &#8212; blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

Mess Nitrit und Nitrat, Metabolite in der Stickoxid-Bahn, in biologischen Materialien der Chemilumineszenz-Methode unter Verwendung von

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

der Anbau von<em&gt; Caenorhabditis elegans</em&gt; In drei Dimensionen im Labor

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Probenvorbereitung und Bildgebung von Exosomen von Transmissions-Elektronenmikroskopie

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Measuring Crop Motility and Food Passaging in Drosophila

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. Disordered GI motility and gastric emptying cause multiple diseases and symptoms. As a powerful genetic model organism, Drosophila can be used in GI motility research. The Drosophila crop is an organ that contracts and moves food into the midgut for further digestion, functionally similar to a mammalian stomach. Presented is a protocol to study Drosophila crop motility using simple measurement tools. A method for counting crop contractions to evaluate crop motility and a method for detecting the distribution of food dyed blue between the crop and gut using a spectrophotometer to investigate the effect of the crop on food passaging is described. The method was used to detect the difference in crop motility between control and nprl2 mutant flies. This protocol is both cost-efficient and highly sensitive to crop motility.

Measuring Crop Motility and Food Passaging in Drosophila

The goal of this protocol is to measure crop contraction and quantify food distribution in the Drosophila gut.

Messung der Pflanzenmotilität und Lebensmittelpassierung in Drosophila

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. ...