Os autores gostariam de agradecer a H. Olivares e D. Ruge para discussões e conselhos valiosos. Esta pesquisa foi apoiada em parte por um prêmio NSF SBIR (IIP-1127245 para BioSentinel Inc.) e um contrato do Departamento de Defesa (W81XWH-07-2-0045 para BioSentinel Inc.).

FM Dunning, TM Piazza, F. Zeytin e WC Tucker são funcionários ou proprietários de BioSentinel Inc. BioSentinel atualmente fabrica e comercializou alguns dos reagentes apresentados neste relatório.

Este protocolo descreve procedimentos para a quantificação de BoNT / A complexa, holotoxina, Clostridium ou de sobrenadante de cultura em matrizes complexas. O protocolo é o mesmo, no entanto, ao testar outros serotipos BoNT (por exemplo, de BoNT / B, E e F), com os seus respectivos ensaios de matriz 56,60, embora a sensibilidade do ensaio irá variar entre os serotipos e ensaios. Este protocolo não conta para cada tipo de amostra possível e algumas modificações que podem ser necessá...

Neurotoxinas botulínicas (BoNTs) são as substâncias mais letais conhecidos, com doses letais humanos intravenosos estimadas em 1-3 ng / kg 1,2. Sete serotipos estruturalmente semelhantes de BoNT, rotulados de A a G, existem, cada um constituído por um domínio de cadeia pesada responsável pela ligação à célula, a absorção e a translocação para o citosol e uma cadeia leve que codifica uma endopeptidase de zinco 3-5. A toxicidade requintado de BoNT resulta, em parte, a sua entrada obrigatória e específica para os neurônios motores na junção neuromuscular 6. Uma vez dentro do neurônio, a endopeptidase cadeia leve cliva especificamente um ou mais do-N-sensível etilmaleimida receptor solúvel da proteína fator de fixação (SNARE) proteínas necessárias para a fusão das vesículas, inibindo a liberação de neurotransmissores e levando a paralisia flácida 7-14. Vulgarmente conhecida como a doença &quot;botulismo&quot;, paralisia do diafragma e músculos intercostais por BoNT finalmente, resulta eminsuficiência respiratória e morte, a menos que o diagnóstico precoce eo tratamento são recebidos. Transmitidas por alimentos Humano botulismo é mais comumente associado com BoNT sorotipos A, B, E e F (BoNT / A, BoNT / B, etc) e, geralmente, resulta da ingestão de alimentos contaminados 15,16, embora, vários casos de botulismo de feridas foram relatados entre os usuários de drogas injetáveis ​​17,18. Nos Estados Unidos, o botulismo infantil resultante da ingestão de esporos de Clostridium por crianças com menos de um ano de idade é a forma mais comum de botulismo 19-21. No entanto, de origem alimentar surtos BoNT resultantes de conservas casa imprópria e processamento de alimentos foram relatados, tanto nos Estados Unidos e no exterior. Entre 2000-2009, pelo menos 338 casos de botulismo alimentar foram relatados em todo o mundo, incluindo seis mortes 22. A capacidade de detectar com rapidez e sensibilidade surtos de botulismo de origem alimentar é uma indicação importante que pode auxiliar no diagnóstico precoce 23,24. Além disso, os métodos de detecção que permitem que o custo-benefício e análise de alimentos de rotina vai levar à melhoria da segurança alimentar. Especificidade neuronal do BoNT e longa meia-vida biológica, também faz com que seja um agente terapêutico potente. Nos Estados Unidos, as drogas à base de BoNT são aprovados pela Food and Drug Administration para o tratamento de condições de cosméticos e perturbações relacionadas com neuromusculares incluindo linhas glabelares, distonia cervical, enxaqueca, bexiga hiperactiva, e estrabismo. Inúmeras aplicações &quot;off-label&quot; são documentados, incluindo tratamentos com altas doses para a disfunção muscular grave 25-28. Accurate quantificação de toxina é fundamental para o doseamento correcto, como subdoses podem conduzir a um tratamento ineficaz, enquanto sobredosagem coloca os doentes em risco de efeitos secundários potencialmente prejudiciais. Infelizmente, nenhum protocolo de teste de potência padronizada é compartilhada entre fabricantes, resultando em desigualdades definição unidade entre produtores de drogas baseado em BoNTcts 29-31. O teste padrão para BoNT é o bioensaio em que BoNT contendo amostras são injetadas por via intraperitoneal em camundongos e os números de óbitos registrados mais de 1-7 dias 16,32,33. O bioensaio é muito sensível com limites de detecção (LOD) de 5-10 pg BoNT / A 34, no entanto, as preocupações éticas sobre o uso de animais, o alto custo de treinamento de pessoal e manutenção de instalações de animais, longos tempos de ensaio, bem como a falta de protocolos padronizados resultou em chamadas para desenvolver padronizado, livre de animal testes BoNT e métodos de quantificação 35-39. Recentemente, vários métodos alternativos de quantificação BoNT foram desenvolvidos que oferecem mouse ou quase-rato sensibilidade bioensaio 40-49. Esses métodos geralmente usam de fluorescência, espectrometria de massa, ou métodos imunológicos e oferecer tempos de ensaio consideravelmente mais curto que o bioensaio em ratos sem uso de animais. Espectrometria de massa combinada com abordagens techniq imunológicaues foram mostrados para detectar e quantificar BoNT contida nos alimentos e outras amostras complexas, no entanto, os requisitos de formação de pessoal e equipamento especializado limite Estes ensaios 50-55. A maioria dos outros ensaios alternativos não são facilmente aplicáveis ​​a testes de amostras complexas ou não têm o rendimento necessário para o teste BoNT rotina. A natureza altamente variável de viscosidade da amostra de alimento, pH, teor de sal, e constituintes da matriz apresenta um desafio especialmente difícil quando tentando desenvolver métodos in vitro de ensaio com sensibilidade para combinar a potência extrema de BoNT. Além disso, mesmo sistemas de tampão simples e relativamente benignas, tais como os que resultam da libertação de medicamentos à base de BoNT, conter sal, albumina, e estabilizadores de açúcar (isto é, excipientes) que ter um impacto significativo na potência in vitro de BoNT 56. Purificação Toxina é necessário para testes atividade precisa de todos, mas a mais simples das amostras 56-59. OBoTest ensaios Matrix foram projetados para rápida, de alta taxa de transferência, e quantificação consistente de BoNT a partir de amostras de alta complexidade, utilizando equipamentos comumente encontrados em laboratórios de pesquisa 56,60. Estes ensaios use esferas paramagnéticas covalentemente ligadas a anticorpos anti-BoNT sorotipo-específicos para ligar e seqüestrar BoNT de uma amostra e, em seguida, remover a interferir compostos de matriz por lavagem. Após a lavagem, a atividade proteolítica limite BoNT é então quantificada em um tampão de reação otimizada usando um repórter compatível com o sorotipo BoNT sendo testado. Estes repórteres são proteínas f luorogénicos que consistem de uma proteína fluorescente ciano N-terminal (PCP) e uma porção proteína fluorescente amarela derivado do C-terminal (Vénus) radical ligado por um substrato de BoNT, os resíduos 141-206 ou os resíduos de SNAP25 sinaptobrevina 33-94 constituindo o BoTest A / E ou B / D / F repórteres / g, respectivamente 45. Repórter clivagem por BoNT é monitorado usando Förster transferência de energia por ressonância (FRET). Quando tO repórter está intacta, excitação do PCP resulta em FRET a Vênus, extinguindo PCP emissão e emissão Vênus emocionante. A clivagem do repórter por BoNT impede FRET, levando a um aumento na emissão de PCP e diminuição da emissão de Vénus. Actividade de BoNT pode então ser medida quantitativamente usando a razão das emissões PCP e Vénus. LOD inferior a 3 pg são possíveis a partir de uma vasta gama de alimentos, utilizando um formato de placa de 96 poços de alto rendimento 56. O aumento de sensibilidade pode ser obtido usando os volumes de amostra maiores uma vez que o ensaio permite a concentração de toxina na superfície do grânulo. Os ensaios Matrix BoTest para BoNTs A, B, E, e F foram desenvolvidos e testados com alimentos, produtos farmacêuticos e amostras ambientais 56,60. Aqui, nós descrevemos processos para a execução destes ensaios para a detecção de BoNT em baixa complexidade (por exemplo, produtos farmacêuticos, BoNT em tampão) e alta complexidade (por exemplo, alimentos, ambiental) amostras. Métodos de processamento específicospara vários tipos de amostras são abordadas neste protocolo e tipos de amostras não descritos aqui geralmente pode ser adaptado usando uma combinação dos métodos apresentados. O protocolo foi desenvolvido e testado com BoNT / A, mas pode ser adaptado para outros serótipos de BoNT utilizando os seus respectivos ensaios, como demonstrado em outros lugares 56,60.

<table border="0" cellpadding="0" cellspacing="0" width="648">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:192px;">BoTest Matrix A Botulinum Neurotoxin Detection Kit</td>
			<td style="width:107px;">BioSentinel</td>
			<td style="width:104px;">A1015</td>
			<td style="width:245px;">Detection kits for BoNT/B and F are also available.</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:192px;">Varioskan Flash fluorescence microplate reader</td>
			<td style="width:107px;">Thermo-Fisher Scientific</td>
			<td style="width:104px;">5250040</td>
			<td style="width:245px;">Most monochromator- or filter-based units with 434 nm excitation and 470 and 526 nm emission capability can be used.</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:192px;">96-well Magnetic Bead Separation Plate</td>
			<td style="width:107px;">V&#38;P Scientific&#160;</td>
			<td style="width:104px;">VP771H</td>
			<td style="width:245px;">Other magnetic plates may be used, but the plate should be designed to separate the beads to the side of the well.</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:192px;">Magnetic Bead-Compatible Plate Washer</td>
			<td style="width:107px;">BioTek&#160;</td>
			<td style="width:104px;">ELx405 VSRM</td>
			<td style="width:245px;">Optional, only required for automated plate washing.&#160; Other magnetic bead-compatible plate washers may also be used, but should be tested before use.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:192px;">Microcentrifuge</td>
			<td style="width:107px;">Various</td>
			<td style="width:104px;">N/A</td>
			<td style="width:245px;">Optional, only required for samples needing centrifugation.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:192px;">MixMate plate mixer</td>
			<td style="width:107px;">Eppendorf</td>
			<td style="width:104px;">22674200</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:192px;">Orbital Shaker</td>
			<td style="width:107px;">Various</td>
			<td style="width:104px;">N/A</td>
			<td style="width:245px;">Used at room temperature or at 25 &#176;C If temperature control is available</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:192px;">EDTA-free Protease Inhibitor Tablets</td>
			<td style="width:107px;">Roche</td>
			<td style="width:104px;">4693132001</td>
			<td style="width:245px;">Only required for food or environmental testing.&#160; Protease inhibitors must be EDTA-free.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:192px;">BoNT/A&#160;</td>
			<td style="width:107px;">Metabiologics</td>
			<td style="width:104px;">N/A</td>
			<td style="width:245px;">Optional, only required for standardization and quantification purposes&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:192px;">Black, Flat-bottomed 96-well Plates</td>
			<td style="width:107px;">NUNC</td>
			<td style="width:104px;">237105</td>
			<td style="width:245px;">Plates should not be treated</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:192px;">96-well Plate Sealing Tape</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:104px;">15036</td>
			<td style="width:245px;"></td>
		</tr>
	</tbody>
</table>

isolation and quantification of botulinum neurotoxin from complex matrices using the botest matrix assays

Detecção e quantificação de neurotoxina botulínica (TB) em matrizes complexas exacta é necessário para produtos farmacêuticos, ambientais e de teste da amostra de alimento. É necessário o teste rápido BoNT de alimentos durante surto forense, o diagnóstico do paciente e testes de segurança de alimentos, enquanto for necessário os testes de potência exata para fabricação de produtos à base de drogas BoNT e segurança do paciente. O bioensaio amplamente utilizado para testes de BoNT é altamente sensível, mas não tem a precisão e rendimento necessário para o teste rápido e rotineiro BoNT. Além disso, o uso do bio-ensaio de animais resultou em apelos de autoridades reguladoras de produtos de drogas e defensores dos direitos dos animais em os EUA e no exterior para substituir o bioensaio em ratos para testar BoNT. Vários ensaios in vitro de substituição têm sido desenvolvidos que funcionam bem com BoNT purificada em tampões simples, mas a maior parte não tem sido mostrado para ser aplicável a ensaios em matrizes altamente complexas. Aqui, um protocolo para a detecção deBoNT em matrizes complexas utilizando os BoTest Matrix ensaios é apresentado. O ensaio consiste em três partes: A primeira parte envolve a preparação das amostras para teste, a segunda parte é um passo de imunoprecipitação utilizando anticorpos anti-BoNT esferas paramagnéticas revestidas de anti-corpo para purificar BoNT a partir da matriz, e a terceira parte quantifica proteolítica da BoNT isolado atividade usando um repórter fluorogênico. O protocolo é escrito para o teste de alto rendimento em placas de 96 poços utilizando matrizes tanto de líquidos e sólidos e requer cerca de 2 hr da preparação manual com tempos totais de doseamento de 4-26 horas, dependendo do tipo de amostra, carga de toxinas, e sensibilidade desejada. Os dados são apresentados para BoNT / D teste com solução salina tamponada com fosfato, um medicamento, o sobrenadante de cultura, de 2% de leite, e os tomates frescos e inclui a discussão de parâmetros críticos para o sucesso do ensaio.

Um diagrama que resume as etapas do protocolo descrito é mostrado na Figura 2. O ensaio requer entre 4-26 horas para ser concluído, dependendo do tipo de amostra e sensibilidade do ensaio desejado, mas apenas ~ 2 horas de hands-on do tempo. O ensaio é realizado em placas de 96 poços e, dependendo do tipo de teste a ser realizado, permite o teste em triplicado de uma amostra de 20, incluindo as normas por placa. A Figura 3 mostra os resultados do ensaio u...

Watch this Scientific Journal Video about Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays at JoVE.com

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

A neurotoxina botulínica BoTest Matrix (BoNT) ensaios de detecção de purificar e quantificar rapidamente BoNT a partir de uma variedade de matrizes de amostra. Aqui, apresentamos um protocolo para a detecção e quantificação de BoNT a partir de matrizes sólidos e líquidos e demonstrar o ensaio com BOTOX, tomate e leite.

Isolamento e Quantificação da neurotoxina botulínica a partir de matrizes complexas usando os BoTest Matrix Ensaios

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...

isolation-quantification-botulinum-neurotoxin-from-complex-matrices

Research

JoVE Journal

Neuroscience

15.9K visualizações.  BioSentinel Inc., Madison, WI.  A neurotoxina botulínica BoTest Matrix (BoNT) ensaios de detecção de purificar e quantificar rapidamente BoNT a partir de uma variedade de matrizes de amostra. Aqui, apresentamos um protocolo para a detecção e quantificação de BoNT a partir de matrizes sólidos e líquidos e demonstrar o ensaio com BOTOX, tomate e leite. 

Vídeo: Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Isolamento e Expansão do Rato Adulto células-tronco neurais utilizando o ensaio Neurosphere

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Neurociência

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Dissecção e Cultura do mouse e dopaminérgico do estriado explantes em três dimensões Ensaios matriz de colágeno

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

Imuno-histoquímica para Topografia Wholemount cerebrais, Complexo

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Isolamento e cultura de células endoteliais da Embryonic posencéfalo

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

Produção e Isolamento de axônios de neurônios sensoriais para análise bioquímica Usando filtros porosos

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.

Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

A alta Ensaio imagem Conteúdo de Identificação da neurotoxina botulínica Inibidores

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

Excitação externa de Neurônios Usando Campos Elétricos e Magnéticos em One e culturas bidimensionais

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

Enumeração de células-tronco neurais Usando clonais Ensaios

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Isolamento e Cultura de células estaminais adultas neurais do Subcallosal Zona Rato

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the cells themselves. Here, we present the method of Light-Induced Molecular Adsorption of Proteins (LIMAP) using the PRIMO system as a patterning technique to produce micro-patterned extracellular matrix (ECM) substrates using a single or combination of proteins. The method enables printing of ECM patterns in micron resolution with excellent reproducibility. We provide a step-by-step protocol and demonstrate how this can be applied to study the processes of neuronal pathfinding. LIMAP has significant advantages over existing micro-printing methods in terms of the ease of patterning more than one component and the ability to generate a pattern with any geometry or gradient. The protocol can easily be adapted to study the contribution of almost any chemical component towards cell fate and cell behavior. Finally, we discuss common issues that can arise and how these can be avoided.

Our overall aim is to understand how cells sense extracellular cues that lead to directed axonal growth. Here, we describe the methodology of Light-Induced Molecular Adsorption of Proteins, used to produce defined micro-patterns of extracellular matrix components in order to study specific events that govern axon outgrowth and pathfinding.

Adsorção molecular induzida por luz de proteínas usando o sistema PRIMO para Micropadronização para estudar as respostas celulares às proteínas da matriz extracelular

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the ...