Este trabalho foi financiado pela Agência de Pesquisa esloveno (projeto J2-9764 e um programa de P2-0249). Este vídeo representa material suplementar para o &quot;Eletroporação baseado em Tecnologias e Tratamentos&quot; workshop científico e pós-graduação, organizado pela Faculdade de Engenharia Elétrica da Universidade de Ljubljana, na Eslovénia.

I. Colocação das células com Cell trackers CMFDA e CMRA <ol><li> Experimentos foram realizados em células de camundongo previamente preparado linhagem de células de melanoma (B16-F1). As células são cultivadas em dois separados 25 cm 2 frascos de cultura (TPP, ZDA) a confluência de 70-80% em meio de cultura DMEM (meio modificado Dulbecco Eagle), suplementado com 10% de soro fetal bovino, 0,15 mg / ml L-glutamina, 16 mg / ml de gentamicina (todos da Sigma-Aldrich, Alemanha), 200 unidades / ml crystacillin (Pliva, Croácia), e incubados em 5% de CO 2 a 37 ° C. </li><li> Prepare duas soluções 10 mM estoque de trackers Cell (Invitrogen, EUA), acrescentando 10,76 mL e 9 mL (para CMFDA Verde e Laranja para CMRA, respectivamente) de DMSO (Sigma-Aldrich, Alemanha) a 50 mg do corante no original frasco Invitrogen. A solução de reserva podem ser armazenadas no frigorífico a 4 ° C por alguns meses. Antes de iniciar os experimentos, aquecer a solução até que os cristais de DMSO dissolver. </li><li> Prepare-bicarbonato livre Krebs-Hepes tampão (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). Em dois tubos de 15 ml Eppendorf separadamente mistura 2,1 mL de cada solução estoque (10 mM CMFDA ou CMRA, respectivamente) em 3 ml de bicarbonato de livre tampão Krebs-Hepes. Isso gera uma &quot;solução de carga&quot;, contendo cerca de 7 mM CMFDA (ou CMRA). </li><li> Lave as células duas vezes com bicarbonato sem tampão Krebs-Hepes e insira o carregamento soluções para os frascos. Incubar as células por 30 minutos em 5% CO 2 a 37 ° C. Durante esta primeira incubação reagentes passar livremente através das membranas celulares, mas uma vez dentro da célula, os reagentes são transformados em células impermeant produtos da reação fluorescente. </li><li> Após a incubação em primeiro lugar, lavar e incubar as células com meio de cultura por mais duas horas em 5% CO 2 a 37 ° C. </li><li> Trypsinize células em ambos os frascos (carregado com CMFDA e CMRA) e misturar as células vermelhas e verdes juntos em uma proporção de 1:1 em um tubo de centrífuga de 50 ml (TPP, ZDA). Ajuste a concentração de células de 5 x 10 6 células / ml por diluição com meio DMEM ou por concentração com a centrífuga. Colocar uma gota 20 l de suspensão de células em cada poço da placa de 24 com vários poços (TPP, ZDA). Incubar as células em 5% de CO 2 a 37 ° C por 20 min para permitir que um pouco anexar à superfície do poço e estabelecer contatos celular. </li></ol> II. Eletrofusão <ol><li> Prepare tampão fosfato de potássio isoosomolar (10 mM KH2PO4, 10 mM K 2 HPO 4, 1mM MgCl 2, 250 mM de sacarose) e tampão fosfato de potássio hypoosmolar (10 mM KH2PO4, 10 mM K 2 HPO 4, 1mM MgCl 2, 75 mM de sacarose) . </li><li> Coloque a vários poços com células para a posição microscópio estágio, os eletrodos no fundo do poço e conectá-los ao gerador de pulsos. </li><li> Lavar as células com 1 ml de tampão fosfato de potássio isoosomolar. Adicionar 350 mL de tampão fosfato de potássio hypoosmolar a fim de induzir inchaço celular. O buffer deve cobrir os eletrodos. </li><li> Deixe células em tampão hypoosmolar por 2 minutos antes da aplicação de impulsos elétricos. Durante este tempo influxo de moléculas de água dentro das células devido ao desequilíbrio osmótico entre o interior eo exterior das células causar um aumento do volume celular. Pulsos elétricos devem ser aplicados quando as células estão perto do seu volume máximo, antes de começar a regulamentar o volume diminui. </li><li> Para alcançar eletrofusão ideal e manter a viabilidade celular, os parâmetros ideais de pulsos elétricos devem ser usados. Estes dependem de linha celular utilizada [1]. Neste experimento um trem de pulsos retangulares de 8 (cada uma com duração de 100 mS a 1 Hz) é aplicado a cada amostra, utilizando dispositivo de eletroporação (no nosso caso Cliniporator, IGEA, Itália). Os pulsos são entregues a duas paralelas eletrodos Pt / Ir fio com 0,8 mm de diâmetro e 5 mm de distância entre eles, criando um campo elétrico de aproximadamente 1200 V / cm entre os eletrodos em cada bem, exceto para o controle de poço. </li><li> Deixam as células sem ser perturbado por 10 minutos após a entrega de pulso. Determine o rendimento de fusão por meio de microscopia de contraste fluorescente e fase. </li></ol> III. Aquisição de imagem e determinação do rendimento de fusão <ol><li> As células são observados usando um microscópio de fluorescência (no nosso caso Zeiss Axiovert 200, Zeiss, Alemanha) equipado com um objetivo (x20) e uma câmera CCD refrigerado (VisiCam 1280, Visitron, Alemanha). As imagens são adquiridas em Metamorph 7.1.1 (dispositivos Molecular, EUA), mas o software de aquisição semelhante também pode ser usado. CMFDA está animado com um monocromador (Polychrome IV, Visitron, Alemanha) a 492 nm e 548 nm no CMRA. A fluorescência de CMFDA e CMRA é adquirido através de dois filtros de emissão, um centrado em 535 nm (HQ535/30m, por CMFDA) e os outros centrada em 510 nm (D605/55m, para CMRA, tanto Chroma, EUA). O uso de espelho dicróico (Q515LP) impediu a conversa entre canais. </li><li> Adquirir três imagens (contraste de fase, fluorescência vermelha e verde) para cinco campos escolhidos aleatoriamente em cada poço. Criar três imagens de cada canal triplet imagem. Na imagem, citoplasma fluorescentes podem ser vistos em conjunto com as membranas celulares. Células fusionadas podem assim ser facilmente determinado [Figura 1]. </li><li> Contar todos os três tipos de células (vermelho, verde e duplamente fluorescente) em cada imagem do canal três. Determinar a porcentagem de células duplamente fluorescentes, dividindo o número de células duplamente fluorescentes com o número de todas as células em cada imagem. Rendimento de fusão é definida como a porcentagem de células duplamente fluorescentes multiplicado por 2 já que metade das células fundidas não são detectadas (quando as células do fusível da mesma cor). </li></ol> Resultados representante <img src="/files/ftp_upload/1991/1991fig1.jpg" alt="Figura 1" /> Figura 1 Três imagem de microscopia canal de B16F1 células após eletrofusão:. Contraste de fase, CMRA de fluorescência (excitação em 548 nm) e CMFDA de fluorescência (excitação a 492 nm), ampliação da objetiva 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>

Não há conflitos de interesse declarados.

A capacidade das membranas celulares para fundir não especificamente, por exemplo, por campos elétricos externos, é importante para a medicina, biotecnologia e pesquisa em biologia. Fusão inespecíficos como permite a produção de grande valor células híbridas e seus produtos, tais como anticorpos monoclonais, e fornece informações sobre os mecanismos fundamentais de fusão [2]. Eletrofusão é um método muito eficaz, já que pode ser devidamente ajustada para diferentes tipos de células. Eletrofusão é alcançado quando as células em contato físico são trazidos para seu estado fusogenic (propenso a fusão), por meio de pulsos de alta tensão elétrica. A eficiência de eletrofusão depende de vários parâmetros que afetam duas partes do processo de eletrofusão. Primeira parte do processo de eletrofusão é realização do contato físico entre as células, que pode ser obtido com diferentes métodos [3-8]. Método de aderência (células de crescimento de confluência) pode ser usado de forma eficiente, devido aos contatos célula espontaneamente estabelecido nas zonas de grande entre as células, no entanto, produz muito grandes células com núcleos fundidos muitos. Estamos usando o método modificado adesão, onde as células mais pequenas (com 2-5 núcleos), que são mais propensos a sobreviver e proliferar, são obtidos (Figura 1). Contato entre as células também se beneficiam com inchaço osmótica das células, devido ao tratamento osmótico utilizado no experimento [9]. Segunda parte do processo de eletrofusão é a realização do Estado fusogenic das membranas celulares. Estado Fusogenic se correlaciona bem com o estado electropermeabilized da membrana (células não são especificamente permeabilizadas de moléculas que normalmente não podem passar através da membrana intacta) e é regida pelos mesmos parâmetros dos pulsos elétricos (amplitude, número, comprimento e freqüência) [10] . Os valores dos parâmetros elétricos necessários para o eletroporação ideal [1] e eletrofusão diferem entre diferentes células e dependem de células tamanho e suas propriedades biológicas. Parâmetros elétricos, portanto, precisam ser otimizadas para diferentes linhagens celulares, que são usados ​​como parceiros de fusão, a obtenção de fusão.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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

Eletrofusão celular é um método seguro e não-virais e não-químicas que podem ser usados ​​para preparar células híbridas para terapia humana. Eletrofusão consiste na aplicação de curto pulsos de alta voltagem elétrica para as células que estão em contato próximo. Aplicação de curto, pulsos de alta tensão elétrica provoca desestabilização da membrana plasmática celular. Membranas desestabilizado são mais permeáveis ​​para moléculas diferentes e também propenso à fusão com qualquer membranas vizinhas desestabilizadas. Eletrofusão é, portanto, um método conveniente para alcançar uma fusão não-específicos de células muito diferentes<em&gt; In vitro</em&gt;. A fim de obter membranas de fusão, celular, desestabilizado por campo elétrico, deve estar em contato próximo para permitir a fusão de suas bicamadas lipídicas e, conseqüentemente, seu citoplasma. Neste vídeo, demonstramos eletrofusão eficiente de células<em&gt; In vitro</em&gt; Por meio do método de adesão modificado. Neste método, as células podem se conectar apenas ligeiramente à superfície do poço, de modo que o meio pode ser trocado e as células conservam a sua forma esférica. Visualização de fusão é avaliada pela rotulagem de pré-citoplasma de células com diferentes corantes fluorescentes rastreador celular; metade das células são rotulados com laranja CMRA ea outra metade com o verde CMFDA. Rendimento de fusão é determinada como o número de células fluorescentes duplamente dividida com o número de todas as células, multiplicado por dois.

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

Cell Electrofusion Visualized with Fluorescence Microscopy

Neste vídeo demonstramos eletrofusão eficiente de células<em&gt; In vitro</em&gt; Por meio do método de adesão modificados utilizando eletroporação ea detecção posterior de visualização células fundidas com microscopia de fluorescência.

Electrosoldável célula visualizado com microscopia de fluorescência

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 visualizações.  University of Ljubljana. Neste vídeo demonstramos eletrofusão eficiente de células In vitro Por meio do método de adesão modificados utilizando eletroporação ea detecção posterior de visualização células fundidas com microscopia de fluorescência.

Vídeo: 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.

Replicação de DNA visualizar Único molécula com microscopia de fluorescência

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

Biologia

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.

Liberação de dopamina no Individual Terminais Pré-Sinápticas visualizado com 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.

Lapso de tempo de imagens de Mitose Depois de Transfection siRNA

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

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.

Quantitativas de células vivas de Análise de microscopia de fluorescência de levedura

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.

Simultânea Multicolor Imagem de Estruturas Biológicas com fluorescência Fotoativação Localização Microscopia

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.

Interações proteína-proteína visualizado por Bimolecular Fluorescência de Complementação em tabaco Protoplastos e folhas

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.

Preparação da amostra para microscopia da força atômica de virion único e imagem de fluorescência de super-resolução

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.

Visualizando Frequency Stromule com microscopia de fluorescência

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

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

Microscopia de Luz Folha de fluorescência das raízes das plantas que crescem na superfície de um gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

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.

Viver a microscopia de fluorescência da célula para observar os processos essenciais durante o crescimento de células microbianas

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