Este trabajo fue apoyado por la Agencia de Investigación de Eslovenia (proyecto J2-9764 y el programa P2-0249). Este video representa el material complementario para la &quot;base de electroporación, tecnologías y tratamientos&quot; taller científico y postgrado, organizado por la Facultad de Ingeniería Eléctrica de la Universidad de Ljubljana, en Eslovenia.

I. Carga de las células con células seguidores CMFDA y CMRA <ol><li> Los experimentos se realizaron en células previamente preparada de la línea celular de ratón melanoma (B16-F1). Las células se cultivan en dos separados 25 frascos de cultivo cm 2 (TPP, ZDA) al 70-80% de confluencia en medio de cultivo DMEM (Dulbecco modificado de Eagle), complementado con suero fetal bovino al 10%, 0,15 mg / ml de L-glutamina, 16 mg / ml de gentamicina (todos de Sigma-Aldrich, Alemania), 200 unidades / ml crystacillin (Pliva, Croacia), y se incubaron en el 5% de CO 2 a 37 ° C. </li><li> Preparar dos soluciones madre 10 mM de seguidores celular (Invitrogen, EE.UU.) mediante la adición de 10,76 l y 9 l (para el verde y CMFDA de Orange CARM, respectivamente) de DMSO (Sigma-Aldrich, Alemania) a 50 mg de colorante en el original Invitrogen vial. La solución madre se pueden almacenar en un refrigerador a 4 º C durante unos pocos meses. Antes de comenzar los experimentos, calentar la solución hasta que los cristales se disuelven de DMSO. </li><li> Prepare bicarbonato sin Krebs-Hepes buffer (130 mM NaCl, 4,7 mM KCl, 1,2 mM MgSO 4, 1,2 mM KH 2 PO 4, 11,7 mM D-glucosa, 1,3 mM CaCl 2, 10 mM HEPES, pH 7,4). En dos de 15 ml tubos Eppendorf separado mezclar 2,1 l de cada solución madre (10 mM CMFDA o CARM, respectivamente) en 3 ml de bicarbonato libre de Krebs-Hepes buffer. Esto produce una &quot;solución de carga&quot;, que contiene aproximadamente 7 M CMFDA (o CARM). </li><li> Enjuague las células dos veces con bicarbonato sin Krebs-Hepes buffer y luego insertar la carga de soluciones en los frascos. Se incuban las células durante 30 minutos en el 5% de CO 2 a 37 ° C. Durante esta primera incubación reactivos pasar libremente a través de las membranas celulares, pero una vez dentro de la célula, los reactivos se transforman en células impermeable productos de reacción fluorescente. </li><li> Después de la incubación primera vez, aclarar e incubar las células con medio de cultivo por otras dos horas en el 5% de CO 2 a 37 ° C. </li><li> Trypsinize células en ambos frascos (cargado con CMFDA y CARM) y la mezcla de glóbulos rojos y verdes, unidos en una proporción de 1:1 en un tubo de centrífuga de 50 ml (TPP, ZDA). Ajustar la concentración de células a 5 x 10 6 células / ml por dilución con medio DMEM o por la concentración de centrífuga. Lugar una caída de 20 l de suspensión celular en cada pocillo de 24 pocillos de la placa (TPP, ZDA). Se incuban las células en el 5% de CO 2 a 37 ° C durante 20 minutos para que puedan colocar un poco a la superficie del pozo y establecer contactos de la célula. </li></ol> II. Electrofusión <ol><li> Prepare el buffer de isoosomolar fosfato de potasio (10 mM KH2PO4, 10 mM K 2 HPO 4, 1 mM MgCl 2, 250 mM de sacarosa) y tampón fosfato de potasio hipoosmolar (10 mM KH2PO4, 10 mM K 2 HPO 4, 1 mM de MgCl2, 75 mM de sacarosa) . </li><li> Coloque el multipocillo con células en el microscopio, la posición de los electrodos en la parte inferior del pozo y conectarlos al generador de pulsos. </li><li> Lavan las células con 1 ml de solución tampón fosfato de potasio isoosomolar. Añadir 350 l de tampón fosfato de potasio hipoosmolar con el fin de inducir la inflamación celular. El tampón debe cubrir los electrodos. </li><li> Salir de las células en tampón hipoosmolar durante 2 minutos antes de la aplicación de pulsos eléctricos. Durante este tiempo de llegada de las moléculas de agua en las células debido a un desequilibrio osmótico entre el interior y el exterior de las células causar un aumento del volumen celular. Impulsos eléctricos se debe aplicar cuando las células están cerca de su volumen máximo, antes de comenzar a disminuir el volumen de regulación. </li><li> Para lograr óptimos de electrofusión y mantener la viabilidad celular, los parámetros óptimos de los impulsos eléctricos se debe utilizar. Estas dependen de la línea celular utilizada [1]. En este experimento, un tren de 8 pulsos rectangulares (cada uno con duración de 100 ms a 1 Hz) se aplica a cada muestra, con dispositivo de electroporación (en nuestro caso Cliniporator, IGEA, Italia). Los pulsos son entregados a dos electrodos paralelos Pt / Ir con un diámetro de 0,8 mm y 5 mm de distancia entre ellos, creando un campo eléctrico de aproximadamente 1200 V / cm entre los electrodos en cada pozo, a excepción del control de pozos. </li><li> Salen de las células en reposo durante 10 minutos después de la entrega de pulso. Determinar el rendimiento de fusión mediante microscopía de contraste de fase y fluorescencia. </li></ol> III. De adquisición de imágenes y la determinación del rendimiento de la fusión <ol><li> Las células se observan con un microscopio de fluorescencia (en nuestro caso Zeiss Axiovert 200, Zeiss, Alemania) equipado con un objetivo (x20) y una cámara CCD enfriado (VisiCam 1280, Visitron, Alemania). Las imágenes se adquieren en MetaMorph 7.1.1 (Molecular Devices, EE.UU.), pero otro software de adquisición similar también se puede utilizar. CMFDA se excita con un monocromador (policroma IV, Visitron, Alemania) a 492 nm y 548 nm en la CARM. La fluorescencia de CMFDA y CMRA se realiza a través de dos filtros de emisión, uno centrado en 535 nm (HQ535/30m, por CMFDA) y el otro centrado en 510 nm (D605/55m, por CMRA, tanto Chroma, EE.UU.). El uso de espejo dicroico (Q515LP) que impidan la diafonía canal. </li><li> La adquisición de tres imágenes (contraste de fases, de color rojo y la fluorescencia verde) durante cinco campos elegidos al azar en cada pozo. Crear tres imágenes de canales de cada triplete de la imagen. En dicha imagen citoplasma fluorescentes pueden verse, junto con las membranas celulares. Células fusionadas por lo tanto se puede determinar fácilmente [Figura 1]. </li><li> Contar con los tres tipos de células (rojo, verde y fluorescente dual) en cada imagen de tres canales. Determinar el porcentaje de células fluorescentes doblemente dividiendo el número de células doblemente fluorescente con el número de todas las células en cada imagen. Rendimiento de fusión se define como el porcentaje de células fluorescentes doblemente multiplicado por 2, ya que la mitad de las células fusionadas no se detectan (cuando las células de la mecha del mismo color). </li></ol> Resultados representante <img src="/files/ftp_upload/1991/1991fig1.jpg" alt="Figura 1" /> Figura 1 Imagen de microscopía tres canales de B16F1 células después de electrofusión:. Contraste de fases, CMRA fluorescencia (excitación a 548 nm) y CMFDA fluorescencia (excitación a 492 nm), aumento del objetivo 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>

La capacidad de las membranas celulares de fusionar de forma no específica, por ejemplo, por campos eléctricos externos, es importante para la biotecnología, la medicina y la investigación en la biología. Fusión inespecíficos, permite la producción de células híbridas de gran valor y sus productos, como los anticuerpos monoclonales, y proporciona información sobre los mecanismos fundamentales de la fusión [2]. Electrofusión es un método potencialmente muy eficaz ya que puede ser ajustado correctamente a diferentes tipos de células. Electrofusión se logra cuando las células en contacto físico se ponen en su estado fusogénica (predisposición a la fusión) por medio de pulsos de alta tensión eléctrica. La eficiencia de electrofusión depende de varios parámetros que afectan a dos partes del proceso de electrofusión. Primera parte del proceso de electrofusión es el logro de un estrecho contacto físico entre las células, que pueden ser obtenidos con diferentes métodos [3-8]. Método de adherencia (las células que crecen a la confluencia) se puede utilizar de manera eficiente gracias a los contactos de manera espontánea de células establecidas en zonas grandes entre las células, sin embargo, que produce células fusionadas muy grandes con muchos núcleos. Estamos utilizando el método de adherencia modificado, en células más pequeñas (de 2 a 5 núcleos), que tienen más probabilidades de sobrevivir y proliferar, se obtienen (Figura 1). Contacto entre las células también se benefician de la hinchazón osmótica de las células, debido al tratamiento osmótico utilizado en el experimento [9]. Segunda parte del proceso de electrofusión es el logro del estado fusogénica de las membranas celulares. Estado fusogénica se correlaciona bien con el estado electropermeabilized de la membrana (células no son específicamente permeabilized a las moléculas que normalmente no pueden pasar a través de la membrana intacta) y se rige por los mismos parámetros de los pulsos eléctricos (amplitud, duración, número y frecuencia) [10] . Los valores de los parámetros eléctricos necesarios para la electroporación óptima [1] y electrofusión difieren entre las distintas células y depende del tamaño de las células y sus propiedades biológicas. Parámetros eléctricos tanto, deben ser optimizados para diferentes líneas celulares, que se utilizan como socios de la fusión, para obtener la fusión.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<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>
</tr>
<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>
<tr>
<td>KCl</td>
<td>Reagent</td>
<td>Merck</td>
<td>A154336 908</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>M2643</td>
<td>&nbsp;</td>
<tr>
<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>
		</div>

cell electrofusion visualized with fluorescence microscopy

Electrofusión de células es un método seguro, no-virales y no químicas que pueden ser utilizados para la preparación de células híbridas para la terapia humana. Electrofusión implica la aplicación de corta pulsos de alta tensión eléctrica a las células que están en contacto cercano. Aplicación de corto y de alta tensión de los impulsos eléctricos provoca la desestabilización de las membranas de las células plasmáticas. Desestabilizado las membranas son más permeables a las moléculas diferentes y también son propensos a la fusión con cualquier membrana vecinos desestabilizado. Electrofusión es, pues, un método conveniente para lograr una fusión no específicos de células muy diferentes<em&gt; In vitro</em&gt;. Con el fin de obtener la fusión, las membranas celulares, desestabilizado por el campo eléctrico, debe estar en estrecho contacto para permitir la fusión de las bicapas de los lípidos y por lo tanto su citoplasma. En este vídeo, se demuestra electrofusión eficiente de las células<em&gt; In vitro</em&gt; Por el método de adherencia modificado. En este método, las células se permite adjuntar sólo un poco a la superficie del pozo, por lo que el medio se pueden cambiar y las células conservan su forma esférica. Visualización de la fusión es evaluado por pre-etiquetado del citoplasma de las células con diferentes tintes fluorescentes célula de seguimiento, la mitad de las células se marcan con color naranja CARM y la otra mitad de verde CMFDA. Rendimiento de fusión se determina el número de células fluorescentes doblemente dividido por el número de todas las células se multiplican por dos.

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

Cell Electrofusion Visualized with Fluorescence Microscopy

En este video se demuestra electrofusión eficiente de las células<em&gt; In vitro</em&gt; Por el método de adhesión modificar mediante electroporación y la posterior detección de la visualización de células fusionadas con microscopía de fluorescencia.

Electrofusión de células visualiza con microscopía de fluorescencia

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 vistas.  University of Ljubljana. En este video se demuestra electrofusión eficiente de las células In vitro Por el método de adhesión modificar mediante electroporación y la posterior detección de la visualización de células fusionadas con microscopía de fluorescencia.

Video: 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.

Visualización de una sola molécula de ADN de replicación con microscopía de fluorescencia

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

Investigación

Biología

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.

La liberación de dopamina en los terminales presinápticos individuales visualizados con 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.

Time-lapse de imágenes de mitosis después de la transfección de siRNA

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.

Basado en la fluorescencia de medición de la entrada de calcio de la tienda que funciona en las células vivas: a partir de células cancerosas cultivadas de fibra muscular esquelética

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.

Cuantitativa de células vivas por fluorescencia microscopia análisis de la levadura de fisión

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.

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.

Interacciones proteína-proteína se visualizó por fluorescencia Bimolecular Complementación en protoplastos de tabaco y hojas

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.

Preparación de muestras para microscopía de fuerza atómica de virión único e imágenes de fluorescencia de súper resolución

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.

La visualización de frecuencia Stromule con microscopía de fluorescencia

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.

Vivo de la célula de microscopía de fluorescencia para observar procesos esenciales durante el crecimiento de la célula microbiana

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