NZ gostaria de agradecer aos membros do laboratório Subhas Biswas, Andrew Sproul, e Ryan Willet para a formação dela em dissecar e colheita neurônios simpáticos. Suportada por concessões do NIH-NINDS.

1. Preparação para a dissecação: <ol><li> Selecione o prato de cultura apropriado (10 cm ou placas de seis poços ou 24 poços, etc), dependendo da natureza de suas experiências, e revesti-los com rat tail-colágeno 24 horas antes da dissecção. Os métodos para a preparação de rato-cauda de colágeno e usá-lo para placas de cultura de revestimento têm sido descritos em outra parte (1). </li><li> Prepare uma solução de tripsina 0,25% em tampão fosfato salino (PBS) (sem EDTA) e filtro de esterilizar. Prepare um alíquotas ml e armazenar a -20 º C. </li><li> Prepare uma solução contendo 2,5 mM de cada Uridina e 5-FDU em água destilada / deionizada H 2 O. Filtro de esterilizar e preparar 50 mL alíquotas. Armazenar a -20 º C. </li><li> Prepare Médio Completo: meio de cultura RPMI 1640 com inativado pelo calor de 10% soro de cavalo (calor inativar incubando em banho-maria 56 º C por 30 minutos) e 5% de soro fetal bovino. </li><li> Prepare Médio Final: meio de cultura RPMI 1640 com inativado pelo calor 1% soro de cavalo + NGF (100ηg/ml concentração final) + penicilina / estreptomicina (1 / 250) + 1 / 250 uridine/5-FDU solução (10 mM de concentração final) . Nota: este meio deve ser composto fresco antes de usar o tempo todo. Para um experimento em um formato de placa de 24 cavidades, a pessoa precisa preparar 12 ml deste meio. É recomendado que você prepare um pouco mais. NGF pode ser comprado de uma variedade de fontes comerciais (ver tabela no final deste protocolo). </li><li> Equipamento: Um vai precisar de um microscópio de dissecação e fonte de luz. Um iluminador de fibra óptica dupla gooseneck com dicas foco é o preferido. Além disso, teremos de limpar e esterilizar (em autoclave ou imersão em etanol 70% por pelo menos 20 minutos) as ferramentas de dissecção seguinte: dois pares de micro-dissecção pinças de aço inoxidável, e um par de tesouras de dissecação (também de aço inoxidável e tanto do Roboz). Duas agulhas de seringa estéril (26 gague x 1 ½ polegadas ou similar) também será necessário. É necessário criar um quadro de dissecção, que é um pedaço 3 &quot;x3&quot; de isopor embrulhado em papel de alumínio e coberto com uma Kimwipe. Pulverizar a placa com álcool 70% para descontaminar. Finalmente, prepare um fogo-polido pipeta de vidro de pastagem. , A fim de-fogo polonês, tome a pipeta de vidro e manter a ponta na chama por alguns segundos, enquanto gira. Após a inspeção, a ponta ficará mais suave ea abertura será mais estreito. Isso é necessário para a etapa onde as células serão dissociados. Executar fogo polimento em uma capa de cultura de células para que permanece a pipeta estéril. </li></ol> 2. Dissecção da Gânglios Cervical Superior: <ol><li> Coletar filhotes de rato recém-nascidos que estão em idade de P0 ou P1. </li><li> Rapidamente limpar o filhote para dissecação com etanol 70% para reduzir as chances de contaminação. </li><li> Rapidamente decapitar filhote com uma tesoura afiada (este passo não será mostrado no vídeo). Segure a cabeça, usando uma pinça estéril contra gaze esterilizada brevemente drenar o excesso de sangue. </li><li> Colocar a cabeça na almofada dissecção com o lado ventral para cima ea extremidade caudal orientada para você. A traquéia cortada deve ser facilmente visível. Use as agulhas de seringa estéril para proteger a cabeça cortada para o bloco de dissecação da seguinte forma: empurre um pino que o céu da boca no teclado e empurre o segundo pino que a traquéia cortada no teclado. Coloque a almofada sob o microscópio de dissecação e começar a dissecção. </li><li> Utilizando ambas as pinças (uma em cada mão), pele clara distância e gordura ao redor da traquéia, tomando cuidado para não ir muito fundo. Fazendo isso irá expor um par de músculos praticamente paralelos entre si em ambos os lados da traquéia. Nota: durante a dissecção, você vai precisar para irrigar com o frio PBS estéril ou frio, isento de soro RPMI 1640, entregues com uma estéril, pipeta Pasteur para lavar o excesso de sangue e para manter a área limpa e úmida. Isso pode ser feito apenas uma vez ou mais de uma vez dependendo de quão rápido se trabalha. Para um dissector experiência, que leva apenas cerca de 2-3 minutos para dissecar um par de gânglios. </li><li> Use a pinça para cortar e remover o músculo de um lado primeiro. </li><li> Uma vez que o músculo é removido, a artéria carótida se tornará visível. Muitas vezes é a luz rosada e contém sangue. Pode parecer cor mais clara que outros vasos sanguíneos que você pode ver na vizinhança e por esta razão a artéria carótida pode não ser notado de imediato. Esta artéria corre ao lado da traquéia e bifurca-se em o que parece um &quot;Y&quot; em forma de fita. Esta bifurcação é um marco importante e uma vez que está localizado, será relativamente fácil encontrar a SCG. </li><li> Sever artéria no final mais caudal e levante-o ligeiramente com a pinça (do outro lado ainda está em anexo). Uma vez que você fizer isso, você vai ver uma forma de amêndoa, massa olhar brilhante pequena de tecido que é vagamente ligado à artéria apenas no ponto de bifurcação e que tem thread-como pré e / ou pós-ganglionares conectivos. Este é o gânglio cervical superior. Use um segundo par de pinças na outra mão para retirá-la com cuidado e coloque em um prato de cultura pequeno (35 mm) contendo frio, isento de soro RPMI 1640. </li><li> Em seguida, extrair o gânglio do outro lado da traquéia seguindo o mesmo protocolo. </li></ol> 3. A limpeza da Gânglios: <ol><li> Uma vez que todos os gânglios foram dissecados, eles devem ser libertados, tanto quanto possível de tecido estranho. </li><li> Sob o microscópio de dissecção, você pode ver que os gânglios ter anexado pedaços de artéria carótida, tecido adiposo ou outros. Trabalho com uma pinça em cada mão para arreliar fora esses materiais indesejados. Lugar limpo gânglios em um estéril 15ml cônicos, tubos de polipropileno, contendo meio RPMI 1640. </li><li> Conectivos pré e / ou pós-ganglionares também deve ser aparado. Cada gânglio tem cerca de 10 mil neurônios. Uma vez que os neurônios são banhados, eles vão regenerar suas neurites. </li></ol> 4. Estabelecer cultura Neuron simpática: <ol><li> Centrifugar os gânglios no 200xg por 2 minutos e desprezar o sobrenadante. </li><li> Ressuspender os gânglios em 0,5 a 1 ml da solução de tripsina 0,25%. Coloque em uma 37 ˚ C banho-maria ou incubadora durante 30 minutos. Isso ajudará a dissociar as células nos gânglios. </li><li> Após 30 minutos, adicione 10 ml de meio completo para diluir-se e neutralizar os tripsina e centrifugar a 200xg por 2 minutos. </li><li> Elimine o sobrenadante e ressuspender em 2 ml de meio final. </li><li> Além disso dissociar as células por trituração dos gânglios cima e para baixo com o fogo-polido pipeta de vidro de pastagem. Triturar os gânglios 20 vezes e colocar o tubo no gelo por alguns minutos. </li><li> Enquanto isso, o fogo-polonês pipeta a mais para fazer a abertura mais estreita (cerca de 2 mm / 3 a 02/01 de diâmetro). Aguarde alguns minutos até que esfrie e triturar os gânglios 20 vezes mais. Como você continuar, o meio se tornará progressivamente cloudier indicando que as células estão a ser dissociados. Repita este passo uma ou duas vezes mais, dependendo de quão bem as células se dissociam. Depois de algum tempo, você vai notar que todos os gânglios têm o dissociada em neurônios e gânglios intactos não são visíveis no tubo. Pode haver algum material de tecido que não se dissociam. Neste ponto, vamos sentar o tubo no gelo por alguns minutos para que todos os detritos não dissociado pode se depositar no fundo. Agora, cuidadosamente, coletar quase todos do meio da parte superior e coloque em outro tubo. Para garantir que você não recolhem os detritos não dissociado, deixar cerca de 50 ul na parte inferior do tubo. Adicionar mais do meio para o final do tubo contendo células dissociadas: este volume depende do tipo de uma placa de cultura está usando para a cultura de neurônios. Por exemplo: se um prato de 24 é bem a ser empregado, então placa 0,5 gânglios por poço (~ 10 mil neurônios por poço) em 0,5 ml de meio de final por poço. Uma ninhada de ratos típico consiste de 12 filhotes, o que daria 24 gânglios. Isso é suficiente para uma placa de sementes de 24 poços eo volume total de meio final é de 12 ml. Nota: os gânglios também contêm uma pequena população de células gliais e outros tipos celulares que estarão presentes na cultura. Usando Uridine/5-FDU no médio final irá impedir a sua proliferação e promover a sua morte. </li><li> Células de alimentação a cada 48 horas, substituindo dois terços do meio RPMI fresco com 1% soro de cavalo + NGF e uridine/5-FDU. Nota: No início, a cultura contém neurônios simpáticos que olhar em volta sem neurites. Neurites curtos tornam visível 24 horas após a dissecção e progressivamente se tornam mais densos e mais ramificadas ao longo do tempo. </li></ol>

<ol>
	<li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , (1998).</li><li>Biswas, S. C., Shi, Y., Sproul, A., Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-apoptotic+Bim+Induction+in+Response+to+Nerve+Growth+Factor+Deprivation+Requires+Simultaneous+Activation+of+Three+Different+Death+Signaling+Pathways.">Pro-apoptotic Bim Induction in Response to Nerve Growth Factor Deprivation Requires Simultaneous Activation of Three Different Death Signaling Pathways.</a> The Journal of Biological Chemistry. 282 (40), 29368-29374 (2007).</li><li>Bocchini, V., Angeletti, P. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nerve+Growth+Factor:+purification+as+a+30,000-molecular-weight+protein.">The Nerve Growth Factor: purification as a 30,000-molecular-weight protein.</a> Proc. Natl. Acad. Sci. USA. (64), 787-794 (1969).</li><li>Dechant, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chat+in+the+Trophic+Web:+NGF+Activates+Ret+by+Inter-RTK+Signaling.">Chat in the Trophic Web: NGF Activates Ret by Inter-RTK Signaling.</a> Neuron. 33 (2), 156-158 (2002).</li><li>Deckwerth, T., Elliott, J. L., Knudson, C. M., Johnson, E. M., Snider, W. D., Korsmeyer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bax+is+required+for+neuronal+death+after+trophic+factor+deprivation+and+during+development.">Bax is required for neuronal death after trophic factor deprivation and during development.</a> Neuron. (17), 401-411 (1996).</li><li>Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+Vitro+Studies+on+the+Nerve+Growth+Factor+(NGF)+Requirement+of+Neurons+-+I.+Sympathetic+Neurons.">Quantitative in Vitro Studies on the Nerve Growth Factor (NGF) Requirement of Neurons - I. Sympathetic Neurons.</a> Developmental Biology. (58), 96-105 (1977).</li><li>Park, D. S., Morris, E. J., Padmanabhan, J., Shelanski, M. L., Geller, H. M., Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin-dependent+kinases+participate+in+death+of+neurons+evoked+by+DNA-damaging+agents.">Cyclin-dependent kinases participate in death of neurons evoked by DNA-damaging agents.</a> The Journal of Cell Biology. 143, 457-467 (1998).</li><li>Pierchala, B. A., Ahrens, R. C., Paden, A. J., Johnson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+Growth+Factor+Promotes+the+Survival+of+Sympathetic+Neurons+through+the+Cooperative+Function+of+the+Protein+Kinase+C+and+Phosphatidylinositol+3-Kinase+Pathways.">Nerve Growth Factor Promotes the Survival of Sympathetic Neurons through the Cooperative Function of the Protein Kinase C and Phosphatidylinositol 3-Kinase Pathways.</a> The Journal of Cell Biology. 279, 27986-27993 (2004).</li></ol>

The authors have nothing to disclose.

Nós usamos ratos Sprague Dawley de nossas culturas. Tire um tempo e cuidado ao limpar os gânglios. Remover todos os detritos, vasos sangüíneos e gordura. Este passo é importante para garantir uma cultura que é mais ou menos homogêneo e livre de tipos de células estranhas. A adição de uridine/5-FDU em grande parte eliminar qualquer resquício de células não-neuronais (mitose) contaminando a cultura ou, pelo menos, reprimir a sua proliferação. Além disso, durante a etapa de dissociação, ser paciente e ter temp...

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<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>Forceps, Stainless steel #5</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS4976</td>
<td>&nbsp;</td>
<tr>
<td>Scissors, DEAVER straight sharp-blunt- 43mm blades - 5.5</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS6760</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640 w/ L-Glutamin</td>
<td>Reagent</td>
<td>Cellgro, Mediatech, Inc.</td>
<td>10-040-CV</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>Reagent</td>
<td>SAFC Biosciences</td>
<td>12103c</td>
<td>&nbsp;</td>
<tr>
<td>Donor Horse Serum</td>
<td>Reagent</td>
<td>JRH Biosciences</td>
<td>12449</td>
<td>Heat-inactivate by incubating in 56&deg;C waterbath for 30 minutes.</td>
</tr>
<tr>
<td>Penicillin/streptomycin</td>
<td>Reagent</td>
<td>Gibco, Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin without EDTA</td>
<td>Reagent</td>
<td>Difco</td>
<td>MT 25-050-CI</td>
<td>&nbsp;</td>
<tr>
<td>Uridine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>U3003</td>
<td>&nbsp;</td>
<tr>
<td>5-Fluoro-5' deoxyuridine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>F-8791</td>
<td>&nbsp;</td>
<tr>
<td>NGF</td>
<td>Reagent</td>
<td>Harlan Bioproduct</td>
<td>BT-5025</td>
<td>&nbsp;</td>
<tr>
<td>Dissection Microscope and light source</td>
<td>Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>15 ml polypropylene tubes</td>
<td>Tool</td>
<td>BD Falcon</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>Cell culture dishes</td>
<td>Tool</td>
<td>Corning, Nunclone</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

protocol for culturing sympathetic neurons from rat superior cervical ganglia (scg)

Os gânglios cervical superior (GCS) de ratos são pequenos, brilhantes, em forma de amêndoa estruturas que contêm neurônios simpáticos. Esses neurônios fornecem inervação simpática para a cabeça e pescoço e constituem uma população bem caracterizados e relativamente homogêneo (4). Neurônios simpáticos são dependentes de fator de crescimento neural (NGF) para a diferenciação, sobrevivência e crescimento axonal ea propagação de grande disponibilidade de NGF facilita sua cultura e manipulação experimental (2, 3, 6). Por estas razões, cultivadas neurônios simpáticos têm sido utilizadas em uma ampla variedade de estudos, incluindo o desenvolvimento neuronal e diferenciação, os mecanismos de morte celular programada e patológicos, e transdução de sinal (1, 2, 5 e 6). Dissecando o SCG de ratos recém-nascidos e neurônios simpáticos cultura não é muito complicado e pode ser dominada com bastante rapidez. Neste artigo, iremos descrever em detalhes como dissecar o SCG de filhotes de rato recém-nascido e para usá-los para estabelecer culturas de neurônios simpáticos. O artigo também descreve as etapas preparatórias e os vários reagentes e equipamentos que são necessários para alcançar este objectivo.

Watch this Scientific Journal Video about Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG at JoVE.com

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

Este é um protocolo que descreve como isolar e cultura primária de neurônios simpáticos gânglios cervical superior (GCS) de filhotes de rato recém-nascido.

Protocolo para Neurônios Cultivar Sympathetic de Gânglios Superior Rat Cervical (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

protocol-for-culturing-sympathetic-neurons-from-rat-superior-cervical

Research

JoVE Journal

Biology

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

22.6K visualizações.  Columbia University. Este é um protocolo que descreve como isolar e cultura primária de neurônios simpáticos gânglios cervical superior (GCS) de filhotes de rato recém-nascido.

Vídeo: Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

Preparando E18 neurônios de rato para Cortical Compartimentação em um dispositivo micro

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

Biologia

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

Isolamento de neutrófilos Protocolo

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

Preparação e manutenção dos neurônios da raiz dorsal Gânglios em culturas Compartmented

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

Uma Introdução ao Lab Worm: de Worms Cultivar a mutagênese

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

Um protocolo simples para extrair Hemócitos de Lagartas selvagens

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

Cultura primária Rat coletor medular interno células dos ductos

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

A amostragem de sangue a partir da veia lateral da cauda do rato

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Isolamento de CD146<sup&gt; +</sup&gt; Células Residente Lung estromais mesenquimais de rato Pulmões

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Um protocolo de alta eficiência simples para isolamento de Islet pancreático de camundongos

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergstr&#246;m technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

This technical report describes a variation of the modified Bergstr&#246;m technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

Coleta de Biópsias Musculares Esqueléticas do Compartimento Superior do Musculus Tibialis Anterior para Avaliação Mecânica

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...