Pesquisa foi suportada pelo NIH Grants EY012031 e EY0182175 ea FM Kirby Foundation.

Pinça óptica para manipulação de células isoladas em cultura Forças aprisionamento de pinças ópticas são gerados a partir do impulso de luz (Ashkin, 1991;. Ashkin et al, 1986). Embora estas forças facilmente armadilha células em suspensão, eles não são capazes de mover as células que aderem a uma superfície. Portanto, para reduzir a adesão ao prato de cultura no ponto onde as células estão presos, a lamela é revestido com poli-2-hydroxyethylmethacrylate (poly-HEMA) (Sigma Chemical Co., St Louis, MO), um composto não tóxico com célula repelente propriedades anteriormente empregados para estudos de adesão em células endoteliais (Folkman e Moscona, 1978). Enquanto poli-HEMA cobre metade da placa de cultura, a outra metade é revestido com um substrato que suporta o crescimento celular. Em nosso laboratório, Sal-1 é usado como substrato para a cultura de células da retina salamandra. O procedimento para a fabricação de Sal-1 e poli-HEMA pratos revestido é descrito abaixo: Fabricação de placas de cultura <ol><li> Esfregaços limpa (# 1 VWR Scientific Inc., Media, PA) em ácido. Esta lamela foi escolhida pela sua espessura de apenas 0,1 milímetros, necessária para a alta objetivos abertura numérica necessário para focar o laser. </li><li> Para criar uma área de repelente de células, dissolver 20 mg de poli-HEMA (Sigma Chemical Co., St Louis, MO) em 1ml de etanol 95% durante a noite a temperatura ambiente. De preferência, esta solução deve ser utilizada dentro de 1 mês de preparação. </li><li> Escorar o esfregaços em uma posição quase vertical em um ambiente livre de poeira, como um capuz. </li><li> Coloque uma ou duas gotas de poli-HEMA solução perto do topo da lamela da ponta de plástico de uma micropipeta 200μl. As gotas (cerca de 50-100μl cada) deve ser restrito à metade da lamela. O declive íngreme garante que o líquido flui para baixo da superfície da lamela para o fundo, proporcionando uma camada fina e uniforme de poli-HEMA. Permitir que o esfregaços poli-HEMA revestido para secar o capô por 30-60 minutos em temperatura ambiente. </li><li> Marcar o lado do poli-HEMA da lamela com uma caneta de ponta fina perto da borda. </li><li> Faça um furo de 1 cm no fundo de um prato de cultura 35 mm. </li><li> Cole a lamela poli-HEMA revestidos para o fundo do prato de cultura com Sylgard 184 (Dow Corning Co., Midland, MI), de modo que o lado poli-HEMA revestido faces em direção ao interior do prato. Garantir a borda do revestimento de poli-HEMA corre abaixo do centro do buraco. Permitir que os pratos para secar por 1-3 dias em temperatura ambiente em um ambiente livre de poeira. </li><li> Riscar uma linha na parte externa da lamínula com uma caneta de ponta de diamante após a borda do revestimento de poli-HEMA. Então, nada duas linhas cruzam em ângulos retos a ele cerca de 2 milímetros de distância. Os dois pontos de interseção servem de referência ou pontos fiducial a ser utilizado para reproduzir a mesma orientação da placa de cultura no palco microscópio. </li><li> Esterilizar os pratos sob luz ultravioleta durante a noite. </li><li> Para criar uma meia-célula de adepto da placa de cultura, o revestimento do meia poli-HEMA não com Sal-1, que contém um rato de anticorpos anti-salamandra levantadas contra as membranas das células da retina (generosamente fornecida pelo Dr. Peter MacLeish, Morehouse School of Medicine , Atlanta, GA; ver MacLeish et al (1983)).. Primeira camada, o Sal-1 lado com cerca de 100μl de 0,1 mg / ml estéril cabra anti-camundongo IgG (Boehringer Mannheim Corporation, Indianápolis IN), tomando cuidado para não derramar o líquido sobre para o lado poli-HEMA. Deixe por três horas, depois removê-lo. Lave a mesma área com solução de Ringer estéril de uma ou duas vezes, em seguida, coloque 100μl Sal-1 sobrenadante, tomando cuidado para não derramar o líquido no lado poli-HEMA do prato. O primeiro anticorpo garante Sal-1 é revestido com uma concentração conhecida. </li><li> Incubar os pratos com o overnight Sal-1. </li><li> Remover a solução Sal-1 anticorpo e lavar a área com solução de Ringer é estéril. Introduzir 2 ml de meio para o prato pouco antes do revestimento celular. </li></ol> A composição da solução Ringer anfíbios, sem soro meio anfíbio, e solução de Ringer para a digestão enzimática são encontradas em MacLeish e Townes-Anderson (1988) e Mandell et al. (1993). Preparação de culturas de células As células utilizadas neste vídeo foram obtidos a partir de luz adaptados, adulto, aquáticos fase salamandras tigre (Ambystoma tigrinum), medindo 17-22 cm de comprimento (Charles Sullivan Inc., de Nashville, TN), mantido a 5 ° C em um 12 h: 12 h ciclo claro-escuro. Os protocolos foram aprovados pelo Institutional Animal Care e Use Committee (IACUC) da Universidade de Medicina e Odontologia de New Jersey em estrita conformidade com as diretrizes do National Institutes of Health. Os procedimentos para a preparação de culturas de células da retina são descritos por Nachman-Clewner e Townes-Anderson (1996) e são as seguintes: <ol><li> Decapitar e medula dos animais. Remove os olhos e dissecar as córneas e íris. O retinas deve separar um pouco dentro da ocular. Ao colocar uma pinça curva debaixo da retina, colher as retinas. Não é importante se a lente está ligado à retina nesta fase. </li><li> Digerir as retinas em 14 U / ml papaína (Worthington, Freehold, NJ) em solução de Ringer com salamandra por 40 minutos. </li><li> Antes do uso, a solução Ringer papaína deve ser aeradas com 95% O 2 / 5% CO 2 gás por 30 minutos. E depois esterilizados por passagem através de um filtro de 0,22 mícron (Millipore, Carrigtwohill, Irlanda). </li><li> Lave as retinas com solução fisiológica de Ringer com salamandra três vezes. </li><li> Remover as lentes de solução de Ringer, em seguida, delicadamente triturar o tecido da retina com um furo de 3 mm pipeta para produzir uma suspensão de células. </li></ol> Laser manipulação de pinças de neurônios isolados <ol><li> Coloque a placa de cultura no palco microscópio, orientando o prato, alinhando uma marca no prato com um ponto de referência no palco. Gravar as coordenadas dos pontos de referência no computador. </li><li> Células chapa para ambos os Sal-1 e poli-HEMA metades do prato. </li><li> Permitir que as células de se contentar com cerca de 20 minutos, que é longo o suficiente para as células do lado aderente do prato para anexar ao substrato de anticorpos. </li><li> Selecione uma célula do lado Sal-1 aderente do prato. Marque o x e estágio y coordenadas de uma posição próxima à célula. No nosso caso, o ponto foi de aproximadamente 10μm a partir dos dendritos primária da célula selecionada. Guardar as coordenadas no computador. </li><li> Selecione um segundo neurônio, no nosso caso um fotorreceptor, no lado poli-HEMA do prato e use a ferramenta de pinça a laser para prendê-lo. Trapping pode ser alcançado através de uma ampla gama de níveis de potência. No entanto, vamos definir o poder do laser em 10% -20%, o que é baixo o suficiente para evitar detritos captura junto com o celular, mas suficiente para transportar o celular através do médium. </li><li> Enquanto segura o celular na armadilha, menor o palco para que a célula está bem acima da superfície da placa de cultura e acima de qualquer neurônios conectados. Mover o palco sob o controle do computador para levar a célula à x salvo anteriormente e coordenadas y no-1 Sal lado. O movimento de estágio foi fixado em 8-20Hm / s, mas a velocidade máxima deve ser determinada empiricamente. À chegada ao ponto de destino no-1 Sal lado, aumentar o palco para levar o celular preso à superfície do prato. Colocação de células pode ser feita com uma precisão de alguns mícrons. Permitem que as células aderem ao substrato Sal-1. </li><li> Obtenção de imagens digitalizadas de ambas as células no par antes e depois da formação de pares fornece um registro útil. </li><li> Manter os pares de células em uma incubadora. Para as células da salamandra, coloque os pratos em uma câmara úmida a 10 ° C. As células podem ser monitorados por lapso de tempo de vídeo e usado com qualquer técnica de células biológicas necessárias. </li></ol>

<ol>
	<li>Ashkin, A., Dziedzic, J. M., Bjorkholm, J. E., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+a+Single-Beam+Gradient+Force+Optical+Trap+for+Dielectric+Particles.">Observation of a Single-Beam Gradient Force Optical Trap for Dielectric Particles.</a> Opt. Lett. 11, 288-290 (1986).</li><li>Clarke, R. J., Hoegnason, K., Brimacombe, M., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cone+and+rod+cells+have+different+target+preferences+in+vitro+as+revealed+by+optical+tweezers.">Cone and rod cells have different target preferences in vitro as revealed by optical tweezers.</a> Mol. Vision. 14, 706-720 (2008).</li><li>Folkman, J., Moscona, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cell+shape+in+growth+control.">Role of cell shape in growth control.</a> Nature. 273, 345-349 (1978).</li><li>Liu, Y., Cheng, D. K., Soneck, G. J., Berns, M. W., Chapman, C. F., Tromberg, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+localized+cell+heating+induced+by+infrared+optical+tweezers.">Evidence of localized cell heating induced by infrared optical tweezers.</a> Biophysical Journal. 68, 2137-2144 (1995).</li><li>MacLeish, P. R., Barnstable, C. J., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+monoclonal+antibody+as+a+substrate+for+mature+neurons+in+vitro.">Use of a monoclonal antibody as a substrate for mature neurons in vitro.</a> Proc. Natl. Acad. Sci. USA. 80, 7014-7018 (1983).</li><li>MacLeish, P. R., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+synapse+formation+among+major+classes+of+adult+salamander+retinal+neurons+in+vitro.">Growth and synapse formation among major classes of adult salamander retinal neurons in vitro.</a> Neuron. 1, 751-760 (1988).</li><li>Mandell, J. W., MacLeish, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Townes-Anderson+E.+Process+outgrowth+and+synaptic+varicosity+formation+by+adult+photoreceptors+in+vitro.">Townes-Anderson E. Process outgrowth and synaptic varicosity formation by adult photoreceptors in vitro.</a> J. Neurosci. 13, 3533-3548 (1993).</li><li>Nachman-Clewner, M., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injury-induced+remodelling+and+regeneration+of+the+ribbon+presynaptic+terminal+in+vitro.">Injury-induced remodelling and regeneration of the ribbon presynaptic terminal in vitro.</a> J. Neurocytol. 25, 597-613 (1996).</li><li>Townes-Anderson, E., St Jules, R. S., Sherry, D. M., Lichtenberger, J., Hassanain, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromanipulation+of+retinal+neurons+by+optical+tweezers.">Micromanipulation of retinal neurons by optical tweezers.</a> Mol. Vis.. 4, (1998).</li></ol>

A luz tem força, e quando um raio de luz é refratada ao passar através de uma célula, uma força é necessária para mudar a direção do momentum. Por causa da lei da conservação do momento, uma força na direção oposta deve, por sua vez, reagem de volta em uma célula. Ashkin (1991) mostrou que a força gerada por um feixe de laser focalizado por uma lente objetiva do microscópio irá mover uma célula para o centro do foco. Mesmo que um feixe de laser gera apenas uma piconewtons alguns de força, essa força é suficiente para p...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>25mm circle No.1 coverglass</td>
<td>&nbsp;</td>
<td>VWR Scientific Inc., Westchester, PA</td>
<td>48380 080</td>
<td>&nbsp;</td>
<tr>
<td>poly-2-hydroxyethylmethacrylate (poly-HEMA)</td>
<td>&nbsp;</td>
<td>Sigma Chemical Co., St Louis, MO</td>
<td>P-3932</td>
<td>Dissolve in 95% ethanol</td>
</tr>
<tr>
<td>Goat anti-mouse IgG antibody</td>
<td>&nbsp;</td>
<td>Chemicon International, Temecula CA</td>
<td>AP181</td>
<td> 1mg in 1ml, dilute 10x for use</td>
</tr>
<tr>
<td>Sal-1 supernatant containing mouse anti-salamander antibody</td>
<td>&nbsp;</td>
<td>generously provided by Dr. Peter MacLeish</td>
<td>&nbsp;</td>
<td>Dr. Peter MacLeish, Morehouse School of Medicine, Atlanta, GA</td>
</tr>
<tr>
<td>3 mm bore 5ml pyrex disposable pipets</td>
<td>&nbsp;</td>
<td>Corning Inc., Corning NY</td>
<td>7078A-5</td>
<td>&nbsp;</td>
<tr>
<td>Cell culture dishes 35mm x 10mm</td>
<td>&nbsp;</td>
<td>Corning inc., Corning NY</td>
<td>430165</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 silicone elastomer kit</td>
<td>&nbsp;</td>
<td>Dow Corning Corp., Midland MI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Optical tweezers-microtool or laser tweezers</td>
<td>&nbsp;</td>
<td>Cell Robotics Inc., Albuquerque NM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 W continuous wave diode laser of 980nm wavelength</td>
<td>&nbsp;</td>
<td>Cell Robotics Inc., Albuquerque NM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Axiovert 100 inverted light microscope</td>
<td>&nbsp;</td>
<td>Carl Zeiss Inc., Thornwood, NY</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>40x oil immersion plan neofluor objective lens</td>
<td>&nbsp;</td>
<td>Carl Zeiss Inc., Thornwood, NY</td>
<td>&nbsp;</td>
<td>Numerical aperture (N.A. 1.3)</td>
</tr>
<tr>
<td>Black and white CCD camera</td>
<td>&nbsp;</td>
<td>Sony Corporation, Tokyo, Japan</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Computer and joystick with software</td>
<td>&nbsp;</td>
<td>Cell Robotics Inc.</td>
<td>&nbsp;</td>
<td>for controlling a motorized stage</td>
</tr>		</tbody>
		</table>
		</div>

using laser tweezers for manipulating isolated neurons in vitro

Neste artigo e vídeo, descrevemos os protocolos utilizados em nosso laboratório para estudar as preferências de segmentação dos processos celulares regeneração de neurônios da retina adulta in vitro. Procedimentos para a preparação de culturas de células da retina começam com a dissecção digestão, e trituração da retina, e terminam com o chapeamento de isoladas as células da retina em pratos feitos especialmente para uso com laser pinças. Estes pratos são divididos em meia célula adesiva e meia repelente celular. O lado adesivo célula é revestida com uma camada de Sal-1 anticorpos, que fornecem um substrato sobre o qual nossas células crescer. Outros substratos adesivo pode ser usado para outros tipos celulares. O lado repelente célula é revestida com uma fina camada de poly-HEMA. As células banhado no lado poli-HEMA do prato são capturados com a pinça a laser, transportado e depois colocado adjacente a uma célula na-1 Sal lado para criar um par. Formação de grupos de células de qualquer tamanho deve ser possível com esta técnica. &quot;Micromanipulação Laser-pinças controlada&quot; significa que o pesquisador pode escolher quais as células a se mover, e a distância desejada entre as células podem ser padronizadas. Porque o feixe de laser atravessa as superfícies transparentes da placa de cultura, seleção e colocação de células são feitas em um ambiente fechado estéril. As células podem ser monitorados por vídeo em tempo-lapso e usado com qualquer técnica de células biológicas necessárias. Esta técnica pode ajudar as investigações de interações célula-célula.

Watch this Scientific Journal Video about Using Laser Tweezers For Manipulating Isolated Neurons In Vitro at JoVE.com

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

Este vídeo descreve a manipulação de neurônios cultivados usando laser pinças in vitro.

Usando uma pinça Laser Para Manipulação neurônios isolados In Vitro

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult ...

using-laser-tweezers-for-manipulating-isolated-neurons-in-vitro

Research

JoVE Journal

Biology

10.3K visualizações.  University of Medicine and Dentistry of New Jersey - UMDNJ. Este vídeo descreve a manipulação de neurônios cultivados usando laser pinças in vitro.

Vídeo: Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

Medição Exocitose em Neurônios Usando Labeling FM

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

Biologia

Silicon Microchips for Manipulating Cell-cell Interaction

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from development to malignant transformation.&nbsp; In particular, interaction with neighboring stroma has been implicated in a plethora of biological phenomena; however, conventional techniques limit the ability to interrogate the spatial and dynamic elements of such interactions.
In Micromechanical Reconfigurable Culture (RC), we employ a micromachined silicon substrate with moving parts to dynamically control cell-cell interactions through mechanical repositioning. Previously, this method has been applied to investigate intercellular communication in co-cultures of hepatocytes and non-parenchymal cells, demonstrating time-dependent interactions and a limited range for soluble signaling 1.
Here, we describe in detail the preparation and use of the RC system. We begin by demonstrating the handling of the device parts using tweezers, including actuating between the gap and contact configurations (cell populations separated by a narrow 80-&micro;m gap, or in direct intimate contact). Next, we detail the process of preparing the substrates for culture, and the multi-step cell seeding process required for obtaining confluent cell monolayers. Using live microscopy, we then illustrate real-time manipulation of cells between the different possible experimental configurations. Finally, we demonstrate the steps required in order to regenerate the device surface for reuse: toluene and piranha cleaning, polystyrene coating, and oxygen plasma treatment.

This article describes an experimental approach for dynamic regulation of cell-cell interactions between adherent cells on a micrometer scale. Manipulation of intercellular communication between hepatocytes and stromal cell is demonstrated. The developed platform enables investigation of cell-cell interactions in a variety of biological processes, including development and pathogenesis.

Microchips de silício para Manipulação de célula-Interação

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from ...

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

Usando Micro-Electro-Mechanical Systems (MEMS) para desenvolver ferramentas de diagnóstico

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Análise de Schwann-astrócito Interações Usando In Vitro Ensaios

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolamento de Ribossomos ligados a polipeptídeos nascentes<em&gt; In vitro</em&gt; Identificar Sites Pausa translacionais Junto mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

In Vitro Agregação ensaios utilizando hiperfosforilada Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

Manipulando o Murino lacrimais Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

A técnica de rim perfundidos rato isolado

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Análise de suspensões celulares isoladas de tecidos sólidos por citometria de fluxo espectral

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.