Financiamento concedido pela National Science Foundation (MF).

Introdução A OMT é uma adaptação de um sistema de corda magnética [1] projetado para incorporar um &quot;simulador de plume virtual&quot;. Os seguintes protocolos irá explicar como moscas corretamente amarrar (Parte 1) e fornecer estratégias para a criação dos ímãs (Parte 2) e de fluxo de massa do sistema de entrega regulada odor (Part3). As informações aqui descrito é otimizado para este sistema em particular e pode variar para componentes com outras especificações técnicas. Parte 1: moscas Tethering Os passos para amarrar aqui descritas são semelhantes a muitos estudos anteriores [2], mas adaptado para uso em um OMT [3]. Tethering adequada é fundamental para garantir robusta e reproduzível ensaios experimentais. A importância de tethering cuidado não pode ser exagerada. <ol><li> Coletar moscas 4-6 dias após a eclosão e privá-los por 4-6 horas. Coloque um kimwipe umedecido com água no frasco desnutridos para evitar desidratação. </li><li> Transferência de um lote de moscas de uma garrafa para um pequeno frasco e inseri-lo em um bloco de bronze em um estágio de resfriamento Peltier fixado em cerca de 4 graus Celsius. A temperatura adequada anestesia as moscas dentro de aproximadamente 30 segundos. </li><li> Classificar as moscas sobre a superfície fria da fase de resfriamento. A Kimwipe úmido garante refrigeração constante e que a absorção de umidade a partir da cutícula. Selecione o maior fêmeas para amarrar. </li><li> Usando um pincel fino, empurre uma única mosca na posição no sarcófago custom-built forma a acomodar um único tipo selvagem mosca da fruta feminina. <img src="/files/ftp_upload/old/1063_2008_11_26_2/1063_Figure-01_thumb.png" alt="" /> Figura 1. <a href="https://www.jove.com/files/1063_2008_10_28_1/1063_Figure-01.png">Por favor, clique aqui para ver uma versão maior da figura do.</a> </li><li> Nós posição um pino minutien (Ferramentas Multas Science) com uma haste magnética montada em um micromanipulador. O pino deve ser alinhado de modo que o fim brusco toca o tórax dorsal logo atrás da cabeça entre as duas alas. O pino deve ser perfeitamente vertical quando visto de frente em direção aos olhos. Mesmo uma pequena quantidade de &#39;roll&#39; entre o pino de voar e não pode ser tolerada. O ângulo de visão lateral entre o pino eo eixo longitudinal do corpo deve estar em um ângulo do nariz para cima de 30 graus em relação ao horizonte (Fig. 1). Nota: Para um funcionamento óptimo no tether ímã, os pinos podem precisar de ser cortado para comprimentos mais curtos. Isso vai depender inteiramente da distância entre os ímãs suspensão. </li><li> Uma vez que o pino minutien está alinhado, use um fio de aço fino para aplicar uma pequena gota de cola UV-ativado (Electrolite Corp) até o fim brusco do pino minutien. </li><li> Manipular o pino para que a gota de cola toca tórax da mosca logo acima da cabeça. Com o tamanho da gota adequada, a cola deve anneal e fluxo do pino sobre o tórax. Tenha cuidado para não usar cola demais. </li><li> Curar a cola com dois de 20 rajadas segundo de luz UV. </li><li> Coloque o fly fixa de lado e repita. Slice pequenos quadrados de Kimwipe e com uma pinça dê um a cada voar amarrado para restringir o vôo. </li><li> Após uma hora de recuperação, as moscas presos estão prontos para experimentos. </li><li> Colagem inadequada pode prejudicar a capacidade de uma mosca para realizar na arena ímã. Cada mosca deve ser verificado para rotação suave no início de cada experimento para garantir as moscas estão devidamente colados. Isso pode ser feito por rotação de um padrão listrado com um período suficientemente grande espacial para obter respostas optomotor forte [4]. Se a mosca é colado no ângulo de passo errado ou inclinado sobre o pino em relação ao eixo de rotação, a mosca não pode (i) giram em tudo, (ii) têm uma gama restrita de movimento, ou (iii) giram em inconsistentes velocidade. Nos casos acima da mosca deve ser descartado e substituído por um novo. Se isso resolver o problema era mais provável um caso de colagem impróprio. (Se substituindo a mosca não resolve o problema, consulte a Parte 2.4) </li></ol> Parte 2: Alinhamento dos ímãs Alinhar os ímãs (Rare-earth-magnets.com) corretamente é necessário que a mosca para alcançar um leque grau suave 360 ​​de movimento. Siga estes passos para alinhar corretamente os ímãs. <ol><li> Os ímãs anel inferior são mantidas horizontalmente por uma luva de plástico e colocado diretamente em cima de uma câmara de vácuo clara (Fig. 1; descrito adiante na parte 3). O imã da haste superior é fixada verticalmente aproximadamente 3 / 4 &quot;acima da ímãs menores. </li><li> Cerca de alinhar os ímãs superiores e inferiores visualmente, colocando o imã da haste superior diretamente acima do ponto central dos magnetos anel inferior. É útil para prender o ímã superior a um micromanipulador (Siskiyou Inc.) para ajuste de escala fina. </li><li> Epóxi tendo uma V-jóia (peças pequenas) sobre a superfície inferior do imã superior para minimizar o atrito rotacional e padronizar o posicionamento voar. </li><li> Coloque uma mosca na arena e ajustar a posição horizontal e vertical posição do imã superior até a mosca pode facilmente e de forma constante girar 360 graus no eixo de guinada. Cada mosca deve ser verificado para rotação suave no início de cada experimento para garantir os ímãs estão devidamente alinhados e que os pinos individuais minutien não estão danificados (ver Parte 1.11). Se os ímãs não estão devidamente alinhados a mosca não pode (i) giram em todas as (ii) têm uma gama restrita de movimento (iii) giram em velocidades inconsistentes. Se o problema for resolvido substituindo a voar com um novo, era mais provável devido a um problema de colagem (veja Parte 1). Se estes problemas existem para todos os voar, é provável que um problema com o alinhamento dos ímãs e ajuste adicional será necessária. </li><li> Atenção: ao jogar com ímãs de terras raras podem ser divertidos, eles são muito perigosos. Uso indevido resulta em equipamentos danificados e / ou danos corporais de menor importância. Agir com extrema cautela com estes ímãs tentadora mas facilmente subestimada. </li></ol> Parte 3: Entrega de Odor <ol><li> O sistema de entrega de odor é baseado em uma descritos anteriormente [5] e é a parte mais difícil para otimizar (Fig. 1). Ela exige muita paciência e de tentativa e erro. Siga estes passos para conseguir rastreamento odor estável. </li><li> O set-up inicial do sistema odor requer a execução do calibre estreito tubo de Teflon (peças pequenas) a partir do gás multiplexador (Sable Systems International) o costume construído água / odor frascos. A saída destes frascos se conecta diretamente aos tubos hipodérmica porta usando o tubo de Teflon mesmo. </li><li> A câmara de vácuo acrílico é posicionado sob a arena para apoiar os ímãs. Nós usamos um tubo de vidro 4 milímetros montado abaixo a mosca ligado à câmara de vácuo para trazer o vácuo de abertura mais perto do fly e reduzir o diâmetro da abertura. </li><li> Coloque uma mosca amarrada na arena com a pinça. </li><li> Ativar um odor atraente (vinagre de maçã funciona bem), e verificar visualmente que a mosca acompanha o odor. Se a mosca parece não ter preferência pelo odor, siga as próximas três etapas. Isso pode exigir várias moscas e vários ensaios. </li><li> Ajustar a posição da porta até que o odor fly parece acompanhar o odor. Montamos as portas odor em micromanipuladores (Siskiyou Inc.) e posicionada dorsal anterior 4 milímetros e 3mm para a cabeça da mosca. </li><li> Ajustar a taxa de fluxo de massa até a mosca parece acompanhar o odor. Nosso meio, são 7ml/min (Sable Systems International MFC-4). </li><li> Ajustar o fluxo do vácuo até que o fly parece acompanhar o odor. Nós definir a taxa de fluxo de vácuo em 13l/min usando um regulador de fluxo (Cole Parmer). Nota: As linhas de construção da entrada de vácuo pode flutuar ao longo do tempo provocando flutuações na estrutura pluma. Pode ser útil para ligar uma bomba de vácuo externo para o regulador de fluxo para as condições de fluxo constante de vácuo. </li><li> Uma vez que a posição da porta e as taxas de fluxo são definidas, periodicamente alternar entre vapor de água e fornecimento de vapor usando um odor comutável usando um multiplexador de gás e verificar visualmente que a mosca só acompanha o odor quando o odor estiver ligado. Ocasionalmente, odor residual no interior do porta odor pode provocar odor de rastreamento quando o odor é desligado. Isso pode ser eliminado sem a remoção da porta odor posicionada corretamente preenchendo os tubos hipodérmica com etanol e coletando-o no final de entrega com um Kimwipe. Uso de ar comprimido, qualquer golpe de etanol restantes para fora e retomar experiências. </li></ol> Resultados representativos: Quando essas etapas forem seguidas corretamente duas coisas devem ser observadas. Primeiro, sob constante rotação estímulos visuais de uma mosca presa colocado na OMT deve girar suavemente 360 ​​graus no eixo de guinada. &quot;Jerky&quot; voltas (chamado sacadas) são características do comportamento de viragem espontânea na ausência de constantes estímulos visuais. Se as moscas não podem rodar sem problemas, aparentemente, colagem impróprio (ver parte 1) e ímãs desalinhado pode ser o culpado (ver Parte 2). Segundo, quando o odor é ligado, a mosca deve cabeça na direção do odor. Quando o odor é desligado, a mosca deve saccade espontaneamente, sem preferência por qualquer posição única na arena. Se as moscas não acompanhar activamente a pluma de odor, a posição da porta de odor, a taxa de fluxo de ar e / ou a taxa de fluxo de vácuo podem precisar de ajustes (ver Parte 3).

<ol>
	<li>Bender, J. A., Dickinson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+stimulation+of+saccades+in+magnetically+tethered+Drosophila.">Visual stimulation of saccades in magnetically tethered Drosophila.</a> J. Exp. Biol. 209, 3170-3182 (2006).</li><li>Tammero, L. F., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collision-avoidance+and+landing+responses+are+mediated+by+separate+pathways+in+the+fruit+fly,+Drosophila+melanogaster.">Collision-avoidance and landing responses are mediated by separate pathways in the fruit fly, Drosophila melanogaster.</a> J. Exp. Biol. 205, 2785-2798 (2002).</li><li>Duistermars, B. J., Frye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossmodal+visual+input+for+odor+tracking+during+fly+flight.">Crossmodal visual input for odor tracking during fly flight.</a> Curr. Biol. 18, 270-275 (2008).</li><li>Reiser, M. B., Dickinson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+display+system+for+insect+behavioral+neuroscience.">A modular display system for insect behavioral neuroscience.</a> J. Neurosci. Methods. 167, 127-139 (2008).</li><li>Frye, M. A., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+output+reflects+the+linear+superposition+of+visual+and+olfactory+inputs+in+Drosophila.">Motor output reflects the linear superposition of visual and olfactory inputs in Drosophila.</a> J. Exp. Biol. 207, 123-131 (2004).</li></ol>

Amarrar moscas e provocando odor rastreamento estável nesta área é relativamente simples. Requer paciência, prática e tentativa e erro. Muitas partes desta arena poderiam ser optimizadas ou substituído para produzir variações no potencial experimental. Por exemplo, versões mais recentes da câmara de vácuo são muito menores e permitir mais espaço abaixo da arena. Além disso, a força eo tipo de terras raras ímãs podem ser variados ligeiramente e portos odor adicionais poderiam ser acrescentados para aumentar o número de tipo...

<!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>Cooling Stage</td>
<td>&nbsp;</td>
<td>Teca</td>
<td>LHP-300CP</td>
<td>&nbsp;</td>
<tr>
<td>Brass Cooling Block</td>
<td>&nbsp;</td>
<td>Custom Machined</td>
<td>&nbsp;</td>
<td>~4 x 8 x 2cm</td>
</tr>
<tr>
<td>Sarcophagus</td>
<td>&nbsp;</td>
<td>Custom Machined</td>
<td>&nbsp;</td>
<td>Contact us for details</td>
</tr>
<tr>
<td>UV-Activated Glue</td>
<td>&nbsp;</td>
<td>Kemxert corp</td>
<td>KOA300</td>
<td>1oz bottle lasts years</td>
</tr>
<tr>
<td>Panel System</td>
<td>&nbsp;</td>
<td>Caltech</td>
<td>http://www.dickinson.caltech.edu/panels</td>
<td>[4]</td>
</tr>
<tr>
<td>Minutien Pins</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-20</td>
<td>&nbsp;</td>
<tr>
<td>Lower Ring Magnets</td>
<td>&nbsp;</td>
<td>Rare-earth-magnets.com</td>
<td>NSN0615</td>
<td>5 - 0.75"o.d. x 0.375" i.d. x 0.125"</td>
</tr>
<tr>
<td>Upper Rod Magnet</td>
<td>&nbsp;</td>
<td>Rare-earth-magnets.com</td>
<td>NSN0750</td>
<td>0.125" x 1"</td>
</tr>
<tr>
<td>V-Jewel Bearing</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>VJ-0469-01</td>
<td>&nbsp;</td>
<tr>
<td>MFC-4 Flow Regulators</td>
<td>&nbsp;</td>
<td>Sable Systems International</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gas Multiplexer</td>
<td>&nbsp;</td>
<td>Sable Systems International</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Teflon Tubing</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>STT-20-C</td>
<td>&nbsp;</td>
<tr>
<td>20 Gauge Hypodermic Tubes</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>HTX-20R-06</td>
<td>&nbsp;</td>
<tr>
<td>Glass Tubing</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>4mm o.d.</td>
</tr>
<tr>
<td>Vacuum Flow Regulator</td>
<td>&nbsp;</td>
<td>Cole Parmer</td>
<td>EW-32464-52</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a magnetic tether system to investigate visual and olfactory mediated flight control in drosophila

Ficou claro por muitos anos que os insetos usam pistas visuais para estabilizar a sua posição em um fluxo de vento. Muitos animais faixa odores transportados pelo vento. Como tal estabilização, visual de rastreamento diretamente contra o vento auxilia no acompanhamento odor. Mas sinais olfativos influenciam diretamente o comportamento acompanhamento visual de forma independente a partir de pistas de vento? Além disso, a recente avalanche de pesquisas sobre a neurofisiologia e genética neurocomportamental de olfato em Drosophila tem motivado cada vez mais tecnicamente sofisticados e quantitativa ensaios comportamentais. Aqui, nós modificamos um sistema de corda magnética originalmente concebido para experimentos visão, equipando a arena com estreitas laminar plumas odor de fluxo. Uma mosca está colado um pino de aço pequenas e suspensas em um campo magnético que lhe permite yaw livremente. Pequeno diâmetro plumas odor de alimentos são direcionados para baixo sobre a cabeça da mosca s, provocando rastreamento estável por uma mosca com fome. Aqui vamos nos concentrar na mecânica crítica de tethering, alinhando os ímãs, a elaboração da pluma de odor, e confirmando rastreamento odor estável.

Watch this Scientific Journal Video about A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila at JoVE.com

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

Aqui nós descrevemos como amarrar uma mosca em um aparelho olfativo tether-magnética (OMT). Descrevemos como alinhar os ímãs de terras raras e portos odor, e como definir as taxas de fluxo de massa, tanto para o estímulo de entrega e sucção a vácuo para alcançar rastreamento odor ideal.

Um sistema de acoplamento magnético para Investigar controle de vôo visual e olfativo Mediated em Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

a-magnetic-tether-system-to-investigate-visual-olfactory-mediated

Research

JoVE Journal

Biology

11.2K visualizações.  University of California, Los Angeles. Aqui nós descrevemos como amarrar uma mosca em um aparelho olfativo tether-magnética (OMT). Descrevemos como alinhar os ímãs de terras raras e portos odor, e como definir as taxas de fluxo de massa, tanto para o estímulo de entrega e sucção a vácuo para alcançar rastreamento odor ideal.

Vídeo: A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

Visualmente Rastreamento Odor Mediated Durante o Voo em Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

Biologia

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Gravações de uma única célula de sucção de cones fotorreceptores rato

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

Uma Abordagem Prática para mapeamento genético induzível Fate: Um Guia Visual para Mark e Track Células In Vivo</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

Detecção rápida de Homogêneos Ensaios Biológicos Usando o sistema de modulação Biossensoriais Magnetic

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Cromatografia de afinidade lipídica mediada por vesículas com separação de células Magnetic Ativado (LIMACS): um novo método para analisar proteínas lipídios Interação

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi de triagem para identificar fenótipos pós-embrionário em C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Medição quantitativa de invadopodia mediada proteólise da matriz extracelular em Contextos individuais e multicelulares

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

Projeto e Análise do Comportamento de Preferência de Temperatura e seu Ritmo Circadiano em Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Um método melhorado para rápido e preciso de medição de desempenho em vôo<em&gt; Drosophila</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

A validação de um modelo de camundongo para Disrupt Complexos LINC de um modo específico de célula

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...