Gli autori desiderano ringraziare H. Olivares e D. Ruge per le discussioni importanti e consigli. Questa ricerca è stata sostenuta in parte da un premio NSF SBIR (IIP-1.127.245 a BioSentinel Inc.) e del Dipartimento della Difesa contratto (W81XWH-07-2-0045 a BioSentinel Inc.).

FM Dunning, TM Piazza, F. Zeytin e WC Tucker sono dipendenti o proprietari di BioSentinel Inc. BioSentinel attualmente produce e ha commercializzato alcuni dei reagenti presentati in questo rapporto.

Questo protocollo descrive le procedure per la quantificazione BoNT / A complesso, holotoxin, o Clostridium cultura surnatante in matrici complesse. Il protocollo è lo stesso, però, quando prova diversi sierotipi di BoNT (ad es BoNT / B, E e F) con i rispettivi saggi Matrix 56,60, anche se la sensibilità del test varia di tutti i sierotipi e saggi. Questo protocollo non tiene conto di ogni tipo di campione possibile e alcune modifiche può essere richiesto a seconda della composizione del...

Neurotossine botuliniche (BoNT) sono le sostanze più letali noti, con via endovenosa dosi letali umane stimate a 1-3 ng / kg 1,2. Sette sierotipi strutturalmente simili di BoNT, etichettati A a G, esistere, ciascuno costituito da un dominio catena pesante responsabile cella vincolante, assorbimento e traslocazione nel citosol e una catena leggera che codifica una endopeptidasi zinco 3-5. La tossicità squisita BoNT deriva da, in parte, la sua voce specifica di legame e in motoneuroni a livello della giunzione neuromuscolare 6. Una volta all&#39;interno del neurone, la endopeptidasi catena leggera specificamente fende uno o più dei solubile N-etilmaleimmide sensibile proteina recettore fattore di attacco (SNARE) proteine ​​necessarie per la fusione delle vescicole, inibendo il rilascio dei neurotrasmettitori e portando alla paralisi flaccida 7-14. Comunemente conosciuta come la malattia &quot;botulismo&quot;, paralisi del diaframma e muscoli intercostali di BoNT in ultima analisi,insufficienza respiratoria e morte a meno che la diagnosi precoce e il trattamento vengono ricevuti. Origine alimentare umana botulismo è più comunemente associato con BoNT sierotipi A, B, E e F (BoNT / A, BoNT / B, ecc) e solitamente deriva dalla ingestione di alimenti contaminati 15,16, anche se, diversi casi di botulismo della ferita sono stati segnalati tra gli utenti tossicodipendenti per via endovenosa 17,18. Negli Stati Uniti, botulismo infantile derivante dall&#39;ingestione di spore di Clostridium dai bambini sotto l&#39;anno di età è la forma più comune di botulismo 19-21. Tuttavia, focolai di origine alimentare BoNT derivanti da conserviera casa improprio e trasformazione dei prodotti alimentari sono stati riportati sia negli Stati Uniti e all&#39;estero. Tra il 2000-2009, di almeno 338 casi di botulismo alimentare sono stati segnalati in tutto il mondo tra cui sei decessi 22. La capacità di rilevare rapidamente e sensibilmente focolai di botulismo di origine alimentare è un&#39;indicazione importante che potrebbe aiutare la diagnosi precoce 23,24. Inoltre, i metodi di rilevamento che consentono di costo-efficacia e analisi degli alimenti di routine porteranno a migliorare la sicurezza alimentare. Specificità neuronale di BoNT e lunga emivita biologica rende anche un potente terapeutico. Negli Stati Uniti, i farmaci a base di BoNT sono approvati dalla Food and Drug Administration per il trattamento di condizioni cosmetiche e disturbi neuromuscolari legati comprese le linee glabellari, distonia cervicale, emicranie, vescica iperattiva, e strabismo. Numerose applicazioni &quot;off-label&quot; sono documentati, compresi i trattamenti ad alte dosi per gravi disfunzioni muscolo 25-28. Accurate tossina quantificazione è fondamentale per il corretto dosaggio, come sottodosaggio può portare a un trattamento inefficace mentre sovradosaggio mette i pazienti a rischio di effetti collaterali potenzialmente dannosi. Purtroppo, nessun protocollo di dosaggio potenza standard è condiviso tra i produttori, con conseguente disparità di definizione dell&#39;unità tra produttori farmaco a base di BoNTcts 29-31. Il test standard per BoNT è il biotest sui topi in cui BoNT contenenti campioni vengono iniettati per via intraperitoneale in topi e il numero di morti registrati oltre 1-7 giorni 16,32,33. Il biotest sui topi è molto sensibile, con limiti di rilevabilità (LOD) di 5-10 pg BoNT / A 34, tuttavia, le preoccupazioni etiche sull&#39;uso degli animali, l&#39;alto costo della formazione del personale e manutenzione degli impianti di polizia, i tempi di analisi lunghi, e la mancanza di protocolli standardizzati portato nelle chiamate a sviluppare standard, privo di animali BoNT prove e metodi di quantificazione 35-39. Recentemente, diversi alternativi metodi di quantificazione BoNT sono stati sviluppati che offrono mouse o quasi il mouse sensibilità bioassay 40-49. Questi metodi usano comunemente fluorescenza, spettrometria di massa, o metodi immunologici e offrono tempi di analisi molto più breve dei biotest sui topi senza l&#39;uso degli animali. Spettrometria di massa approcci combinati con Techniq immunologicaUES hanno mostrato di rilevare e quantificare BoNT contenuta negli alimenti e altri campioni complessi, tuttavia, le esigenze di formazione del personale e limite di attrezzature specializzate questi saggi 50-55. La maggior parte degli altri test alternativi non sono facilmente applicabili a una prova a campione complessa o non hanno la velocità necessaria per l&#39;analisi di routine BoNT. La natura altamente variabile della viscosità del campione alimentare, pH, contenuto di sale, e costituenti della matrice rappresenta una sfida particolarmente difficile quando si cerca di sviluppare metodi in vitro test con la sensibilità per abbinare l&#39;estrema potenza di BoNT. Inoltre, anche i sistemi tampone semplici e relativamente benigni, come quelli derivanti dalla risospensione dei prodotti farmaceutici a base di BoNT, contengono sale, albumina, zucchero e stabilizzanti (cioè eccipienti) che hanno un impatto significativo in vitro BoNT potenza 56. È necessaria purificazione tossina per il test dell&#39;attività accurata di tutti, ma il più semplice dei campioni 56-59. IlBoTest saggi Matrix sono stati progettati per una rapida, high-throughput, e la quantificazione coerente del BoNT da campioni altamente complessi che utilizzano attrezzature che si trovano comunemente nei laboratori di ricerca 56,60. Questi saggi usano perline paramagnetiche covalentemente legati ad anticorpi sierotipo-specifici anti-BoNT di legare e sequestrare BoNT su un campione e poi rimuovere composti interferenti matrice mediante lavaggio. Dopo il lavaggio, legato attività proteolitica BoNT viene quindi quantificata in un tampone di reazione ottimizzato utilizzando un giornalista compatibile con il sierotipo BoNT in fase di test. Questi giornalisti sono proteine ​​fluorogeniche costituiti da una proteina fluorescente ciano N-terminale (PCP) frazione e un C-terminale giallo fluorescente derivato proteico (Venere) frazione collegate da un substrato BoNT, residui SNAP25 141-206 o residui sinaptobrevina 33-94 costituisce il BoTest A / E o B / D / giornalisti F / G, rispettivamente, 45. Reporter scissione da BoNT è controllata mediante Förster trasferimento di energia di risonanza (FRET). Quando tegli giornalista è intatto, eccitazione del CFP si traduce in FRET a Venere, tempra emissione CFP ed emozionante emissione di Venere. Clivaggio del reporter da BoNT impedisce FRET, portando ad un aumento delle emissioni CFP e diminuzione delle emissioni Venus. Attività BoNT può essere misurato quantitativamente utilizzando il rapporto tra le emissioni PCP e Venus. LOD di sotto del 3 pg sono possibili da una vasta gamma di alimenti utilizzando un formato piastra a 96 pozzetti high-throughput 56. Maggiore sensibilità può essere ottenuta usando campioni di volume poiché il test permette concentrazione della tossina sulla superficie delle sfere. I saggi Matrix BoTest per BoNT A, B, E e F sono stati sviluppati e testati con il cibo, farmaceutica e campioni ambientali 56,60. Qui, descriviamo le procedure per l&#39;esecuzione di questi test per la rilevazione di BoNT a bassa complessità (ad es farmaceutica, BoNT in tampone) e elevata complessità (ad esempio alimenti, ambientale) campioni. Metodi di lavorazione specificiper diversi tipi di campioni sono affrontati in questo protocollo e tipi di campioni qui non descritti possono solitamente essere adattati utilizzando una combinazione dei metodi presentati. Il protocollo è stato sviluppato e testato con BoNT / A, ma è adattabile ad altri sierotipi BoNT utilizzando i rispettivi saggi come dimostrato altrove 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

È richiesto il rilevamento preciso e la quantificazione di neurotossina botulinica (BoNT) in matrici complesse per il settore farmaceutico, ambientale e test campione alimentare. È necessario un rapido BoNT sperimentazione di prodotti alimentari durante forense epidemia, la diagnosi del paziente, e la prova di sicurezza alimentare, mentre è richiesto il test di potenza accurate per la fabbricazione di prodotti a base di droga-BoNT e la sicurezza del paziente. Il biotest sui topi ampiamente usato per il test BoNT è altamente sensibile, ma manca la precisione e la velocità necessarie per un rapido e di routine test BoNT. Inoltre, l&#39;uso del saggio biologico degli animali ha portato a inviti da parte delle autorità di regolamentazione dei prodotti di droga e sostenitori dei diritti degli animali negli Stati Uniti e all&#39;estero per sostituire il biotest sui topi per il test BoNT. Diversi pezzi in vitro sono stati sviluppati che funzionano bene con purificata BoNT nel buffer semplici, ma la maggior parte non hanno dimostrato di essere applicabile a test in matrici molto complesse. Qui, un protocollo per la rilevazione diBoNT in matrici complesse utilizzando le BoTest Matrix test è presentato. Il saggio consiste di tre parti: la prima parte prevede la preparazione dei campioni per le prove, la seconda parte è un passo immunoprecipitazione con anticorpi anti-BoNT paramagnetici branelli rivestiti con anticorpo per purificare BoNT dalla matrice, e la terza parte quantifica proteolitica isolato di BoNT all&#39;attività utilizzando un giornalista fluorogenica. Il protocollo è scritto per prove ad alta velocità in piastre a 96 pozzetti utilizzando matrici sia liquidi che solidi e richiede circa 2 ore di preparazione manuale con tempi totali dosaggio di 4-26 ore a seconda del tipo di campione, carico tossina, e la sensibilità desiderata. I dati sono presentati per BoNT / A test con soluzione salina tamponata con fosfato, un farmaco, supernatante di coltura, 2% latte e pomodori freschi e comprende discussione dei parametri critici di successo dosaggio.

Un diagramma che riassume i passaggi del protocollo descritto è illustrato nella Figura 2. Il test richiede tra 4-26 ore per completare a seconda del tipo di campione e dalla sensibilità del test desiderato, ma solo ~ 2 ore di hands-on tempo. Il test viene eseguito in piastre da 96 pozzetti e, a seconda del tipo di test in corso, permette test triplicato di fino a 20 campioni anche standard per piastra. La figura 3 mostra i risultati del test rappresentati...

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

La neurotossina botulinica BoTest Matrix (BoNT) test di rilevazione rapida purificano e quantificare BoNT da una gamma di matrici. Qui vi presentiamo un protocollo per l&#39;individuazione e la quantificazione delle BoNT da matrici sia solide e liquide e dimostriamo il test con BOTOX, i pomodori e il latte.

Isolamento e quantificazione della neurotossina botulinica da matrici complesse usando il BoTest Matrix saggi

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 Views.  BioSentinel Inc., Madison, WI.  La neurotossina botulinica BoTest Matrix (BoNT) test di rilevazione rapida purificano e quantificare BoNT da una gamma di matrici. Qui vi presentiamo un protocollo per l'individuazione e la quantificazione delle BoNT da matrici sia solide e liquide e dimostriamo il test con BOTOX, i pomodori e il latte. 

Video: 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 ed espansione della cellule staminali neurali del mouse utilizzando il saggio neurosfere

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 ...

Ricerca

Neuroscienze

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.

Dissezione e Cultura del mouse dopaminergici e striatali Espianti di saggi di collagene matrice tridimensionale

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.

Immunoistochimica Wholemount per rivelare Topografia Cervello Complex

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 coltura di cellule endoteliali dal embrionali Forebrain

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.

Produzione e isolamento degli assoni dai neuroni sensoriali per l&#39;analisi biochimica Uso dei filtri porosi

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.

Un alto dosaggio Imaging contenuti per l&#39;identificazione di neurotossina botulinica inibitori

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.

Eccitazione esterna dei neuroni Usando campi elettrici e magnetici in mono e culture bidimensionali

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.

Conteggio di cellule staminali neurali L&#39;utilizzo clonali Assays

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 la coltura di adulti neurali cellule staminali dal Subcallosal Zona mouse

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.

Adsordimento molecolare indotto dalla luce delle proteine utilizzando il sistema PRIMO per micro-patterning per studiare le risposte cellulari alle proteine a matrice extracellulare

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