Diese Arbeit wurde von der slowenischen Research Agency (Projekt J2-9764 und das Programm P2-0249) unterstützt. Dieses Video stellt ergänzendes Material für die &quot;Elektroporation basierenden Technologien und Behandlungsmethoden&quot; wissenschaftlichen Workshop und Aufbaustudium, von der Fakultät für Elektrotechnik an der University of Ljubljana, Slowenien organisiert.

I. Das Laden der Zellen mit Cell-Tracker CMFDA und CMRA <ol><li> Die Experimente wurden auf vorher vorbereiteten Zellen der Maus-Melanom-Zelllinie (B16-F1) durchgeführt. Die Zellen werden in zwei getrennten 25 cm 2 Kulturflaschen (TPP, ZDA) auf 70-80% Konfluenz in DMEM Kulturmedium (Dulbecco modifiziertem Eagle-Medium) mit 10% fötalem Rinderserum, 0,15 mg / ml L-Glutamin, 16 mg ergänzt gewachsen / ml Gentamycin (alle von Sigma-Aldrich, Deutschland), 200 Einheiten / ml crystacillin (Pliva, Kroatien) und inkubiert in 5% CO 2 bei 37 ° C. </li><li> Bereiten Sie zwei 10 mM Stammlösungen von Cell-Tracker (Invitrogen, USA) durch Zugabe von 10,76 ul und 9 ul (für Grüne CMFDA und für Orange CMRA jeweils) DMSO (Sigma-Aldrich, Deutschland) bis 50 ug des Farbstoffes in der ursprünglichen Invitrogen Durchstechflasche. Die Stammlösung kann in einem Kühlschrank bei 4 ° C für wenige Monate gelagert werden. Vor Beginn der Experimente, warm up der Lösung, bis sich die Kristalle von DMSO auflösen. </li><li> Bereiten Bicarbonat-freien Krebs-Hepes-Puffer (130 mM NaCl, 4,7 mM KCl, 1,2 mM MgSO 4, 1,2 mM KH 2 PO 4, 11,7 mM D-Glucose, 1,3 mM CaCl 2, 10 mM HEPES, pH 7,4). In zwei 15 ml Eppendorf-Röhrchen getrennt Mix 2,1 ul jeder Stammlösung (10 mM CMFDA oder CMRA jeweils) in 3 ml Bicarbonat-freien Krebs-Hepes-Puffer. Dies ergibt eine &quot;loading solution&quot; mit etwa 7 uM CMFDA (oder CMRA). </li><li> Spülen Sie die Zellen zweimal mit Bicarbonat-freien Krebs-Hepes-Puffer und fügen Sie dann loading Lösungen in den Kolben. Inkubieren Zellen für 30 Minuten in 5% CO 2 bei 37 ° C. Während dieser ersten Inkubation Reagenzien frei durch Zellmembranen, aber einmal im Inneren der Zelle werden die Reagenzien in Zell-impermeante fluoreszierende Reaktionsprodukte umgewandelt. </li><li> Nach der ersten Inkubation, spülen und inkubieren Zellen mit Kulturmedium für weitere zwei Stunden in 5% CO 2 bei 37 ° C. </li><li> Trypsinize Zellen in beiden Flaschen (beladen mit CMFDA und CMRA) und mischen rote und grüne Zellen in einem Verhältnis 1:1 in einem 50 ml Zentrifugenröhrchen (TPP, ZDA). Passen Zellkonzentration auf 5 x 10 6 Zellen / ml durch Verdünnung mit DMEM-Medium oder durch Konzentration mit Zentrifuge. Legen Sie eine 20 pl Tropfen Zellsuspension in jede Vertiefung von 24 Multiwellplatte (TPP, ZDA). Inkubieren Zellen in 5% CO 2 bei 37 ° C für 20 min, damit sie leicht auf der Oberfläche des gut befestigen und zu etablieren Zell-Kontakten. </li></ol> II. Elektrofusion <ol><li> Bereiten isoosomolar Kaliumphosphatpuffer (10 mM KH2PO4, 10 mM K 2 HPO 4, 1 mM MgCl 2, 250 mM Saccharose) und hypoosmolar Kaliumphosphatpuffer (10 mM KH2PO4, 10 mM K 2 HPO 4, 1 mM MgCl 2, 75 mM Saccharose) . </li><li> Legen Sie die Multiwell mit Zellen auf dem Mikroskoptisch, Position der Elektroden an der Unterseite des Brunnens und verbinden sie mit dem Impulsgenerator. </li><li> Waschen Sie die Zellen mit 1 ml isoosomolar Kaliumphosphatpuffer. Add 350 ul hypoosmolar Kaliumphosphatpuffer um Zellschwellung zu induzieren. Der Puffer sollte sich auf den Elektroden. </li><li> Lassen Sie Zellen in hypoosmolar Puffer für 2 Minuten vor dem Anlegen von elektrischen Impulsen. Während dieser Zeit Zustrom von Wasser-Moleküle in die Zellen durch osmotischen Ungleichgewicht zwischen dem Inneren und dem Äußeren der Zellen zu einem Anstieg des Zellvolumens. Elektrische Impulse sollten angewendet werden, wenn Zellen in der Nähe ihrer maximalen Volumina werden, bevor regulatorischen Volumenabnahme starten. </li><li> Um eine optimale Elektrofusion erreichen und aufrechtzuerhalten Lebensfähigkeit der Zellen, sollten die optimalen Parameter der elektrischen Impulse genutzt werden. Diese auf Zelllinie verwendet [1] ab. In diesem Experiment ein Zug von 8 Rechteckimpulse (jeweils mit einer Dauer von 100 us bei 1 Hz) ist zu jeder Probe aufgebracht, mittels Elektroporation Gerät (in unserem Fall Cliniporator, IGEA, Italien). Die Impulse werden auf zwei parallel Pt / Ir-Draht-Elektroden mit 0,8 mm Durchmesser und 5 mm Abstand zwischen ihnen ausgeliefert, der ein elektrisches Feld von etwa 1200 V / cm zwischen den Elektroden in jedem gut, außer für die Steuerung gut. </li><li> Lassen Sie die Zellen ungestört 10 Minuten nach dem Puls-Lieferung. Bestimmen Sie die Fusionsausbeute mittels Fluoreszenz-und Phasenkontrast-Mikroskopie. </li></ol> III. Bildaufnahme und die Bestimmung des Fusionsausbeute <ol><li> Die Zellen werden beobachtet mit einem Fluoreszenzmikroskop (in unserem Fall Zeiss Axiovert 200, Zeiss, Deutschland) mit einem Objektiv (x20) und eine gekühlte CCD-Kamera (VisiCam 1280, Visitron, Deutschland) ausgestattet. Die Bilder werden in MetaMorph 7.1.1 (Molecular Devices, USA) erworben, aber auch andere ähnliche Software zur Erfassung kann ebenfalls verwendet werden. CMFDA ist mit einem Monochromator (Polychrome IV, Visitron, Deutschland) bei 492 nm und CMRA bei 548 nm angeregt. Die Fluoreszenz der CMFDA und CMRA erworben wird mit zwei Emissionsfilter, einer bei 535 nm zentriert (HQ535/30m für CMFDA) und die andere zentriert bei 510 nm (D605/55m, für CMRA, sowohl Chroma, USA). Die Verwendung von dichroitischen Spiegel (Q515LP) verhindert den Kanal Übersprechen. </li><li> Erwerben Sie drei Bilder (Phasenkontrast, rote und grüne Fluoreszenz) für fünf zufällig ausgewählte Felder in jedem gut. Erstellen Sie drei Kanal-Bilder aus jedem Bild Triplett. In einem solchen Bild fluoreszierenden Zytoplasma kann zusammen mit den Zellmembranen gesehen werden. Fusionierten Zellen können somit einfach bestimmt werden [Abb. 1]. </li><li> Zählen Sie alle drei Arten von Zellen (rot, grün und dual fluoreszierend) in jeweils drei Kanal-Bild. Bestimmen Sie den Prozentsatz der dual fluoreszierenden Zellen, indem die Zahl der dual fluoreszierenden Zellen mit der Anzahl aller Zellen in jedem Bild. Fusion Ausbeute ist definiert als der Anteil der dual fluoreszierenden Zellen mit 2 multipliziert definiert, da die Hälfte der fusionierten Zellen nicht erkannt werden (wenn die Zellen in der gleichen Farbe Sicherung). </li></ol> Repräsentative Ergebnisse <img src="/files/ftp_upload/1991/1991fig1.jpg" alt="Abbildung 1" /> Abbildung 1 Drei-Kanal-Aufnahme von B16F1 Zellen nach Elektrofusion:. Phasenkontrast, CMRA Fluoreszenz (Anregung bei 548 nm) und CMFDA Fluoreszenz (Anregung bei 492 nm), Objektiv-Vergrößerung 20x

<ol>
	<li>&#268;emazar, M., Jarm, T., Miklav&#269;i&#269;, D., Ma&#269;ek-Lebar, A., Ihan, A., Kopitar, N. A., Ser&#353;a, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+electric-field+intensity+on+electropermeabilization+and+electrosensitivity+of+various+tumor-cell+lines+in+vitro.">Effect of electric-field intensity on electropermeabilization and electrosensitivity of various tumor-cell lines in vitro.</a> Electro. Magnetobiol. 17, 263-272 (1998).</li><li>Trontelj, K., Reber&#353;ek, M., Kandu&#353;er, M., &#269;urin &#353;erbec, V., Miklav&#269;i&#269;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+bulk+cell+electrofusion+in+vitro+for+production+of+human-mouse+heterohybridoma+cells.">Optimization of bulk cell electrofusion in vitro for production of human-mouse heterohybridoma cells.</a> Bioelectrochemistry. 74, 124-129 (2008).</li><li>Rols, M. -. P., Teissi&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+electrically+induced+permeabilization+and+fusion+of+Chinese+hamster+ovary+cells+by+osmotic+pressure.">Modulation of electrically induced permeabilization and fusion of Chinese hamster ovary cells by osmotic pressure.</a> Biochemistry. 29, 4561-4567 (1990).</li><li>Neil, G., Zimmermann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrofusion.">Electrofusion.</a> Methods in Enzymology. 220, 174-196 .</li><li>Abidor, I. G., Li, L. -. H., Hui, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+cell+pellets:+II.+Osmotic+properties,+electroporation,+and+related+phenomena:+Membrane+interactions.">Studies of cell pellets: II. Osmotic properties, electroporation, and related phenomena: Membrane interactions.</a> Biophysical journal. 67, 427-435 (1994).</li><li>Jaroszeski, M. J., Gilbert, R., Fallon, P. G., Heller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanically+facilitated+cell-cell+electrofusion.">Mechanically facilitated cell-cell electrofusion.</a> Biophys J. 67 (4), 1574-1581 (1994).</li><li>Ramos, C., Bonefant, D., Teissi&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+hybridization+by+electrofusion+on+filters.">Cell hybridization by electrofusion on filters.</a> Analytical biochemistry. 302, 213-219 (2002).</li><li>Cao, Y., Yang, J., Yin, Z. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+high-throughput+cell+electrofusion+in+a+microelectrode-array+chip.">Study of high-throughput cell electrofusion in a microelectrode-array chip.</a> Microfluid Nanofluid. 5, 669-675 (2008).</li><li>U&#353;aj, M., Trontelj, M., Kandu&#353;er, M., Miklav&#269;i&#269;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+size+dynamics+and+viability+of+cells+exposed+to+hypotonic+treatment+and+electroporation+for+electrofusion+optimization.">Cell size dynamics and viability of cells exposed to hypotonic treatment and electroporation for electrofusion optimization.</a> Radiol. Oncol. 43, 108-119 (2009).</li><li>Teissi&#233;, J., Ramos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+electric+field+pulse+induced+long-lived+permeabilization+and+fusogenicity+in+cell+membranes.">Correlation between electric field pulse induced long-lived permeabilization and fusogenicity in cell membranes.</a> Biophys. J. 74, 1889-1898 (1998).</li></ol>

Die Fähigkeit der Zellmembranen zu unspezifisch, zB durch äußere elektrische Felder, Sicherung ist für die Biotechnologie, Medizin und Forschung in der Biologie wichtig. Solche unspezifischen Fusion ermöglicht die Produktion von hochwertigen Hybrid-Zellen und ihre Produkte, wie monoklonale Antikörper, und liefert Informationen über grundlegende Mechanismen der Fusion [2]. Elektrofusion ist eine potenziell sehr wirksame Methode, da kann es richtig, um verschiedene Arten von Zellen eingestellt werden. Elektrofusion wird erreicht, wenn Zellen in engen körperlichen Kontakt in ihre fusogene Zustand gebracht werden (Gefahr der Fusion) mit Hilfe von Hochspannungs-elektrischen Impulsen. Die Effizienz der Elektrofusion hängt von verschiedenen Parametern, die beiden Teile der Elektrofusion Prozess beeinflussen. Erster Teil des Heizwendel-ist das Erreichen des engen körperlichen Kontakt zwischen den Zellen, die mit verschiedenen Methoden erhalten werden [3-8]. Die Einhaltung Methode (wachsenden Zellen bis zur Konfluenz) können effizient durch spontan entstandene Zellkontakte in großen Zonen zwischen Zellen verwendet werden, jedoch bringt es sehr große fusionierten Zellen mit vielen Kernen. Wir sind mit der modifizierten Einhaltung Methode, wo kleinere Zellen (mit 2 bis 5 Kerne), die eher zu überleben und sich vermehren, gewonnen werden (Abbildung 1). Kontakt zwischen den Zellen auch aus osmotischen Schwellung der Zellen profitieren durch osmotische Behandlung im Experiment [9] verwendet. Zweiter Teil der Elektrofusion Prozess ist das Erreichen der fusogene Zustand der Zellmembranen. Fusogene Zustand korreliert gut mit electropermeabilized Zustand der Membran (Zellen sind unspezifisch permeabilisiert, um Moleküle, die normalerweise nicht durch die intakte Membran passieren können) und wird von den gleichen Parametern der elektrischen Impulse (Amplitude, Länge, Anzahl und Häufigkeit) geregelt [10] . Die Werte der elektrischen Parameter für die optimale Elektroporation benötigt [1] und Elektrofusion zwischen verschiedenen Zellen unterschiedlich und hängen von Zellen Größe und ihrer biologischen Eigenschaften. Elektrische Parameter müssen daher für verschiedene Zelllinien, die als Fusionspartner verwendet werden, die Fusion zu erhalten optimiert werden.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CMRA</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>C34551</td>
<td>Cytosolic fluorescent dye</td>
</tr>
<tr>
<td>CMFDA</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>C7025</td>
<td>Cytosolic fluorescent dye</td>
</tr>
<tr>
<td>DMSO</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>D2650</td>
<td>&nbsp;</td>
<tr>
<td>DMEM </td>
<td>Reagent</td>
<td>Sigma-Aldrich </td>
<td>D5671</td>
<td>Dulbecco's modified Eagle's medium</td>
</tr>
<tr>
<td>Fetal calf serum</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F4135</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>G7513</td>
<td>&nbsp;</td>
<tr>
<td>crystacillin</td>
<td>Reagent</td>
<td>Pliva</td>
<td>625110</td>
<td>antibiotic</td>
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<tr>
<td>gentamicin</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>G1397</td>
<td>antibiotic</td>
</tr>
<tr>
<td>Hepes</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>H0887</td>
<td>&nbsp;</td>
<tr>
<td>KH2PO4</td>
<td>Reagent</td>
<td>Merck </td>
<td>A124873 927</td>
<td>&nbsp;</td>
<tr>
<td>KH2PO4</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>4248</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>M-8266</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>Reagent</td>
<td>Fluka</td>
<td>71382</td>
<td>&nbsp;</td>
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<td>KCl</td>
<td>Reagent</td>
<td>Merck</td>
<td>A154336 908</td>
<td>&nbsp;</td>
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<td>MgSO4</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>M2643</td>
<td>&nbsp;</td>
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<td>D-glucose</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>G8270</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>C4901</td>
<td>&nbsp;</td>
<tr>
<td>sucrose</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>16104</td>
<td>&nbsp;</td>
<tr>
<td>Electric pulse generator</td>
<td>Tool</td>
<td>Igea</td>
<td>&nbsp;</td>
<td>Cliniporator VITAE</td>
</tr>
<tr>
<td>Multiwell plate</td>
<td>Tool</td>
<td>TPP</td>
<td>92424</td>
<td>&nbsp;</td>
<tr>
<td>50 ml centrifuge tube</td>
<td>Tool</td>
<td>TPP</td>
<td>91050</td>
<td>&nbsp;</td>
<tr>
<td>15 ml centrifuge tube</td>
<td>Tool</td>
<td>TPP</td>
<td>91015</td>
<td>&nbsp;</td>
<tr>
<td>25 cm2 culture flask</td>
<td>Tool</td>
<td>TPP</td>
<td>90026</td>
<td>&nbsp;</td>
<tr>
<td>Electrodes</td>
<td>Tool</td>
<td>Custom made</td>
<td>&nbsp;</td>
<td>Pt/Ir</td>
</tr>		</tbody>
		</table>
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cell electrofusion visualized with fluorescence microscopy

Zell Elektrofusion ist eine sichere, nicht-virale und nicht-chemischen Methode, die für die Vorbereitung Hybridzellen für die menschliche Therapie eingesetzt werden können. Elektrofusion umfasst die Anwendung von kurzen Hochspannungs-elektrische Impulse auf Zellen, die in engem Kontakt sind. Anwendung von kurz-, Hochspannungs-elektrische Impulse bewirkt eine Destabilisierung der Zell-Plasmamembranen. Destabilisierte Membranen sind durchlässig für verschiedene Moleküle und auch anfällig für Fusion mit einer benachbarten destabilisiert Membranen. Elektrofusion ist somit eine bequeme Methode, um eine unspezifische Verschmelzung von sehr verschiedenen Zellen zu erreichen<em&gt; In-vitro-</em&gt;. Um fusion, Zellmembranen, durch elektrisches Feld destabilisiert zu erhalten, muss in einem engen Kontakt sein, damit Zusammenlegung ihrer Lipid-Doppelschichten und damit ihrem Zytoplasma. In diesem Video zeigen wir, effiziente Elektrofusion von Zellen<em&gt; In-vitro-</em&gt; Durch modifizierte Einhaltung Methode. Bei dieser Methode, Zellen dürfen nur leicht anzubringen, um die Oberfläche des gut, so dass mittelfristig ausgetauscht werden können und Zellen noch behalten ihre Kugelform. Fusion Visualisierung wird durch pre-Kennzeichnung von dem Zytoplasma von Zellen mit unterschiedlichen fluoreszierenden Zellen tracker Farbstoffe untersucht, die Hälfte der Zellen sind mit orangen CMRA und die andere Hälfte mit grünen CMFDA gekennzeichnet. Fusion Ausbeute ist definiert als die Anzahl der dual fluoreszierenden Zellen mit der Anzahl aller Zellen durch zwei dividiert bestimmt.

Watch this Scientific Journal Video about Cell Electrofusion Visualized with Fluorescence Microscopy at JoVE.com

Cell Electrofusion Visualized with Fluorescence Microscopy

In diesem Video zeigen wir effiziente Elektrofusion von Zellen<em&gt; In-vitro-</em&gt; Durch modifizierte Einhaltung Methode mittels Elektroporation und der anschließenden Detektion der fusionierten Zellen Visualisierung mit Fluoreszenzmikroskopie.

Zell Elektroschweißmuffe Visualisierte mit Fluoreszenz-Mikroskopie

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

cell-electrofusion-visualized-with-fluorescence-microscopy

Research

JoVE Journal

Biology

14.0K Aufrufe.  University of Ljubljana. In diesem Video zeigen wir effiziente Elektrofusion von Zellen In-vitro- Durch modifizierte Einhaltung Methode mittels Elektroporation und der anschließenden Detektion der fusionierten Zellen Visualisierung mit Fluoreszenzmikroskopie.

Serie: Cell Electrofusion Visualized with Fluorescence Microscopy

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Visualizing Einzel-Molekül-DNA-Replikation mit Fluoreszenz-Mikroskopie

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Forschung

Biologie

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

Hui Zhang and Niko G. Gubernator contributed equally to this work.

A new means to measure neurotransmission optically using fluorescent dopamine analogs.

Freisetzung von Dopamin an den einzelnen präsynaptischen visualisiert FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Zeitraffer-Imaging der Mitose Nach siRNA-Transfektion

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

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

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

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

Fluoreszenz-basierte Messung von Calcium-Shop-Eintrag in lebenden Zellen: aus kultivierten Krebszelle zu Skelettmuskelfaser

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

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Quantitative Live Cell Fluoreszenz-Mikroskopie Analyse von Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Simultane Multicolor-Imaging biologischer Strukturen mit Fluoreszenzphotoaktivierung Lokalisationsmikroskopie

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Protein-Protein-Wechselwirkungen durch Bimolekulare Fluoreszenz-Komplementation in Tabak-Protoplasten-und Blätter-Visualisierte

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Probenvorbereitung für Single Virion Atomic Force Microscopy und Super-Resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Visualizing Stromulus Frequenz mit Fluoreszenzmikroskopie

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

Live Cell Fluoreszenzmikroskopie um wesentliche Vorgänge während der mikrobiellen Zellwachstum beobachten

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...