D. Sames graças A Harold G. &amp; Leila Y. Mathers Fundação de Caridade e Iniciativas da Universidade de Columbia em Ciência e Engenharia. D. Sames e D. Sulzer agradecer à Fundação McKnight para as inovações tecnológicas em McKnight Prêmio Neuroscience D. Sulzer graças NIDA, NIMH, e os Picower e Fundações da doença de Parkinson. Graças H. Zhang NARSAD. RH Edwards graças a Michael J. Fox Foundation, a National Parkinson Foundation, NIDA e NIMH. Agradecemos a Robert Burke por 6-OHDA injeções e conselho, Mark Sonders para discussão útil, Merek Siu para imagens de análise, programação e Schmoranzer Jan de apoio técnico com a configuração de microscopia TIRF.

Este método foi utilizado na pesquisa, publicada na <a href="http://www.sciencemag.org/cgi/content/full/324/5933/1441">Gubernator et al. Ciência 324 (5933). 1441-1444 (2009)</a> . 1. Preparando aguda fatias estriatais <ol><li> Antes de preparar fatias estriatal aguda, deve-se oxigenar o líquido cefalorraquidiano artificial (ASCF) e chill-lo com gelo por pelo menos 15 minutos antes da extração de cérebro. Manter o frio do gelo ACSF e oxigenado durante a dissecção. ACSF (em mM): NaCl 125, KCl 2.5, NaHCO 3 26, CaCl 2 2,4, MgSO4 1,3, KH 2 PO 4 0,3, glicose 10, HEPES 5; pH 7,3-7,4, 290-295 mOsm. </li><li> Decapitar rato macho sem anestesia. </li><li> Extrair todo o cérebro do rato, e montá-la na bandeja vibratome colocados no vibratome usando Krazy Glue ®. Extração de cérebro e de montagem deve ser feita dentro de 2 minutos. Durante este tempo, certifique-se para manter o frio do gelo ACSF e oxigenado para manter os tecidos saudáveis. </li><li> Corte coronal fatias do cérebro estriado na espessura 250μm bregma entre 1,54-0,62 1. Três fatias inteiras serão obtidos que podem então ser cortada em 6 fatias meia com uma agulha. </li><li> Incubar as fatias de cérebro em ACSF oxigenada em temperatura ambiente por pelo menos 1 hora antes de carregá-sonda. Fatias de mostrar o carregamento FFN511 bom e permanecer ativo para geração de imagens de até 4 horas após a preparação fatia. </li></ol> 2. Carregando FFN511 <ol><li> Prepare FFN511 solução de carga (10μM FFN511 em ACSF); também preparar 100μM ADVASEP-7 em ACSF. Todas as soluções devem ser preparadas de fresco e oxigenado pelo menos 15 minutos antes do embarque para as fatias. </li><li> Individualmente incubar fatia com FFN511 solução de carga por 30 minutos em temperatura ambiente. </li><li> Em seguida, retire o corante ligado ao tecido extracelular incubando a fatia carregada em oxigenado 100μM ADVASEP-7 ACSF por 30 minutos em temperatura ambiente 2. Fatias estão agora prontos para a imagem latente. </li></ol> 3. Imagem FFN511 no fatias de cérebro Imagem de FFN511 nos cortes é realizada utilizando multifotônica microscópio de varredura a laser. Estamos usando um Prairie Ultima multifotônica microscópio de varredura por laser (Estávamos usando uma Zeiss LSM 510 NLO multifotônica microscópio de varredura a laser antes e alguns resultados foram obtidos com o microscópio Zeiss). <ol><li> Coloque a fatia FFN511-carregado na câmara de gravação (RC-27L, Warner) e superfuse com ACSF oxigenado a um fluxo de 1-2 ml por minuto. Em seguida, coloque uma harpa de platina com cordas de nylon em cima da fatia para minimizar o movimento durante o experimento. Para minimizar ainda mais o movimento, permitir que a fatia para estabilizar por pelo menos 10 minutos na câmara antes da imagem. </li><li> Visualizar e localizar o striatum dorsal, com luminescência campo brilhante sob a objetiva de 10x imersão em água. </li><li> Lugar e posição do bipolar torcida eléctrodos de tungsténio na região se realizar um experimento de estimulação. A área de imagem ideal está localizado dentro de 300 mm entre as duas pontas do eléctrodo bipolar estimulante. Coloque esta região no centro do campo visual. </li><li> Imagem FFN511 marcado terminais estriatal em um (0,9 NA) O objectivo da água por ultravioleta 63x de imersão. FFN511 está animado com 760 nm com um laser de Mai Tai Física Spectral e fluorescência ideal de terminais estriatal é visto através de um filtro passa-banda (480-520 nm). As imagens são capturadas no formato de 12-bit com 75 x 75 mM regiões de interesse em 512 pixels de resolução x 512. Estimulado para experimentos de descoloração, para compensar a mudança do eixo z, z uma série de imagens 07/05, separados por 1 mícron no plano z, é obtido para cada período de tempo. </li></ol> 4. FFN511 Descoloração A etiqueta pode ser descorados FFN511 quer por alta concentração de KCl (70 mM usamos KCl), (+)-anfetamina sulfato (AMPH, 20 mM), ou estimulação elétrica. No experimento de descoloração KCl, quando uma solução ACSF alta de potássio é aplicado à fatia do cérebro, o FFN511 destains label dentro de 2 minutos. Registro xyz-t imagens para acompanhar FFN511 descoloração ao longo do tempo. A seguir descreve estimulação elétrica dependente de descoloração terminal dopamina: <ol><li> Para acompanhar FFN511 descoloração ao longo do tempo, xyz-t as imagens são gravadas. Para garantir movimento mínimo da fatia (menos do que 4 mm no eixo z) e não fotobranqueamento pelo laser, controle de imagens de séries temporais do z-stack deve ser obtido por pelo menos 5 minutos antes da estimulação. Caso contrário, ajustar a potência do laser. </li><li> Após essas imagens de controle, continuam a imagem xyz-t, enquanto começam a estimulação nas freqüências de 1, 4 ou 20 Hz. Estímulos em 1, 4 ou 20 Hz (300 mS x 1 mA) são aplicados para o estriado localmente por um isolador de estímulo Iso-Flex acionada por um gerador de Mestre-8 de pulso (Ampi, Jerusalém, Israel) através de eletrodos bipolares. Para minimizara variação da despolarização dos locais de lançamento, temos usado um protocolo de estimulação aplicando 150% da intensidade de estimulação máxima determinada por voltametria cíclica. </li><li> Usar o software adequado para análise de imagem. Em nosso laboratório, usamos Image J (Wayne Rosband, National Institutes of Health, Rockville, MD) e custom-written software em IDL (Research Systems, Boulder, CO) para quantificar a fluorescência puncta e as mudanças com Time3. </li></ol> Resultados Represenative A Figura 1 mostra o carregamento na FFN511 fatia estriatal está concluída. Imagens representativas utilizando objetiva de 10x e 60x são mostrados na Fig.1. A rotulagem seletiva de terminais de dopamina pode ser descorados por 20 mM de anfetamina. A Figura 2 mostra de descoloração de rotulagem FFN511 por KCl elevado. A Figura 3 mostra descoloração de rotulagem FFN511 por estimulação elétrica local. <img src="/files/ftp_upload/1562/1562fig1.jpg" alt="Figura 1" /> Figura 1 FFN511 rótulos terminais de dopamina no estriato-cortical viver fatias aguda (A) de rotulagem, pela FFN511 em aguda córtico-estriatal fatia ao vivo:.. Rotulagem abundante no corpo estriado (STR), rotulagem esparsa no córtex (CTX), e sem rótulo no corpo caloso (CC). Barra de escala:. 100 mm (B) Descoloração de FFN511 do striatum por anfetamina. Painéis da esquerda: antes de anfetaminas; painéis direita: após 20 minutos de 20 de anfetamina mM. Barra de escala: 10 mM. <img src="/files/ftp_upload/1562/1562fig2.jpg" alt="Figura 2" /> Figura 2. Descoloração de FFN511 rotulagem por KCl elevado. Rotulagem FFN511 é descorados dentro de 2 min de aplicação de 70 mM KCl em ACSF. Barra de escala: 10μm. <img src="/files/ftp_upload/1562/1562fig3.jpg" alt="Figura 3" /> Figura 3. Dependente da freqüência de descoloração de rotulagem em FFN511 striatum. (A) estimulação local em 4 Hz resultou na descoloração dos terminais. Estimulação começou em t = 0. Barra de escala: 5 mm (B) Descoloração de FFN511 a 4 Hz é Ca 2 + - e dependente da freqüência.. Controles não receberam estimulação (153 puncta de 3 fatias). Descoloração com cloreto de cádmio (200 mM) foi idêntico aos controles não estimuladas (475 puncta de 5 fatias). As curvas de descoloração para cada freqüência de estimulação foram ajustados por uma função de decaimento exponencial simples e meia-vida (t 1 / 2) valores calculados como τ x 0,693 (1 Hz: 765 puncta de 9 fatias, 4 Hz: 410 puncta de 7 fatias, 20 Hz: 416 puncta de 6 fatias). Consulte a micrografia de vídeo de dois fótons de microscopia de FFN511 descoloração durante exocitose numa preparação estriado cerebral fatia.

<ol>
	<li>Franklin, K. B. J., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxi coordinators. , (1997).</li><li>Kay, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+synaptic+activity+in+intact+brain+and+slices+with+FM1-43+in+C.+elegans,+lamprey,+and+rat.">Imaging synaptic activity in intact brain and slices with FM1-43 in C. elegans, lamprey, and rat.</a> Neuron. 24, 8098-8117 (1999).</li><li>Bamford, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterosynaptic+dopamine+neurotransmission+selects+sets+of+corticostriatal+terminals.">Heterosynaptic dopamine neurotransmission selects sets of corticostriatal terminals.</a> Neuron. 42, 653-653 (2004).</li></ol>

The authors have nothing to disclose.

Neste vídeo, demonstramos um método de visualizar usando análogos opticamente neurotransmissão da dopamina fluorescente. FFN511 é a primeira geração de FFNs temos desenvolvido. Embora tenha sido projetado visando o transportador vesicular neuronal monoamina (VMAT2), que transporta neurotransmissores monoamina do citoplasma em vesículas sinápticas e, especificamente, etiquetas terminais de dopamina no estriado e catecolaminas presumido e / ou terminais de serotonina no córtex (como mostrado na Ciência papel), um período de carga adequado é fundamental para a especificidade desde FFN511 é relativamente hidrofóbica. Descobrimos que a incubação por mais de 40 minutos irá resultar em coloração inespecífica extensa em fatias estriatais. A especificidade pode ser diretamente determinada pela descoloração usando uma alta concentração de KCl, o que deve provocar a libertação de FFN funcional dentro de vesículas sinápticas. Exposição da fatia para FFN511 para menos de 15 minutos irá resultar em sinal fluorescente fraco devido à carga insuficiente do corante nos terminais. Neste protocolo, usamos 100μM ADVASEP-7 para remover o corante ligado ao tecido extracelular. Este passo não é necessário, mas se ele for omitido, o tempo de washout em ACSF precisa ser prolongada. Em resumo, dependendo da preparação que você escolheu, você precisará modificar o período de concentração e de carga para determinar rotulagem ideal.

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>FFN511 (8-(2-Amino-ethyl)-2,3,5,6-tetrahydro-1H,4H-11-oxa-3a-aza-benzo[de]anthracen-10-one)</td>
<td>&nbsp;</td>
<td>Dalibor Sames&#x2019;s laboratory at Columbia University</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ADVASEP-7 </td>
<td>&nbsp;</td>
<td>CyDex, Overland Park, KS</td>
<td>AR-OA7-005</td>
<td>&nbsp;</td>
<tr>
<td>RC-27L Recording chamber</td>
<td>&nbsp;</td>
<td>Warner Instrument</td>
<td>64-0375</td>
<td>&nbsp;</td>
<tr>
<td>PELCO PrepEze 6-Well Holder</td>
<td>&nbsp;</td>
<td>Ted Pella, Inc.</td>
<td>36157-1</td>
<td>For slice incubation</td>
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<td>Master-8 pulse stimulator and Iso- Flex stimulus isolator</td>
<td>&nbsp;</td>
<td>AMPI, Jerusalem, Israel</td>
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dopamine release at individual presynaptic terminals visualized with ffns

O sistema nervoso transmite sinais entre neurônios através de liberação de neurotransmissores durante a fusão das vesículas sinápticas. Para observar a absorção e liberação de neurotransmissores a partir de terminais pré-sinápticos individuais diretamente, nós projetamos fluorescentes neurotransmissores falsa como substratos para o transportador das vesículas sinápticas monoamina. Usando estas sondas para liberar imagem de dopamina no estriado, fizemos várias observações pertinentes a plasticidade sináptica. Descobrimos que a fração de vesículas sinápticas liberando o neurotransmissor por estímulo foi dependente da freqüência de estímulo. A cineticamente distintas &quot;reserva&quot; da população das vesículas sinápticas não foi observada nessas condições experimentais. A heterogeneidade dependente da freqüência de terminais pré-sinápticos foi revelado que era dependente, em parte, os receptores de dopamina D2, indicando um mecanismo para dependente da freqüência de codificação da seleção pré-sináptica. Hui Zhang e Niko Gubernator G. contribuíram igualmente para este trabalho.

Watch this Scientific Journal Video about Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs at JoVE.com

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

Um novo meio de medir a neurotransmissão opticamente usando análogos de dopamina fluorescente.

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 ...

dopamine-release-at-individual-presynaptic-terminals-visualized-with

Research

JoVE Journal

Biology

24.7K visualizações.  Columbia University. Um novo meio de medir a neurotransmissão opticamente usando análogos de dopamina fluorescente.

Vídeo: Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Carregando Drosophila terminais nervosos com indicadores de cálcio

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Biologia

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Rotulagem e hESCs hMSCs com óxido de ferro nanopartículas para Não-invasiva em Rastreamento vivo com ressonância magnética

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Rotulagem células-tronco com corantes fluorescentes para detecção não invasiva com imagens ópticas

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Principal Dissociated Midbrain dopamina culturas de células de recém-nascidos de roedores

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Murine Model for Parkinson&#039;s Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

Modelo murino para a doença de Parkinson: a partir de 6-OH lesão dopamina para teste comportamental

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

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 ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

iCLIP - Transcriptoma-wide Mapeamento das Interações Proteína-RNA com a Resolução Nucleotide Individual

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Preparação Individual Drosophila Ovos Câmaras de imagens ao vivo

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

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 ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

Imagens de fluorescência com um nanômetros Precisão (Fiona)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...