La ricerca è stata sostenuta da sovvenzioni NIH EY012031 e EY0182175 e la FM Kirby Foundation.

Pinzette ottiche per la manipolazione di cellule isolate in coltura Forze cattura di pinzette ottiche sono generati dalla quantità di moto della luce (Ashkin, 1991;. Ashkin et al, 1986). Anche se queste forze facilmente intrappolare le cellule in sospensione, non sono in grado di spostare le cellule che aderiscono a una superficie. Pertanto, per ridurre l&#39;adesione al piatto cultura nel punto in cui sono intrappolati cellule, il vetrino è rivestito con poli-2-idrossietilmetacrilato (poli-HEMA) (Sigma Chemical Co., St. Louis, MO), un composto tossico con cella repellente proprietà precedentemente impiegato per gli studi di adesione sulle cellule endoteliali (Folkman e Moscona, 1978). Mentre poli-HEMA copre la metà del piatto cultura, l&#39;altra metà è rivestita con un substrato in grado di supportare la crescita cellulare. Nel nostro laboratorio, Sal-1 è usato come substrato per la coltura di cellule della retina salamandra. La procedura per la realizzazione di Sal-1 e piatti rivestiti di poli-HEMA è descritto di seguito: Fabbricazione di piatti della cultura <ol><li> Vetrini puliti (# 1 VWR Scientific Inc., Media, PA) in acido. Questo coprioggetti è stato scelto per il suo spessore di appena 0,1 millimetri, necessaria per gli obiettivi ad alta apertura numerica necessaria per mettere a fuoco il laser. </li><li> Per creare una zona repellente cellula, sciogliere 20 mg di poli-HEMA (Sigma Chemical Co., St. Louis, MO) in 1 ml di etanolo al 95% durante la notte a temperatura ambiente. Preferibilmente, la soluzione deve essere utilizzato entro 1 mese di preparazione. </li><li> Sostenere il vetrini in una posizione quasi verticale in un ambiente privo di polvere, come un cappuccio. </li><li> Appoggiare una o due gocce di soluzione di poli-HEMA vicino alla parte superiore del coprioggetti dalla punta di plastica di una micropipetta 200μl. Le gocce (circa 50-100μl ciascuno) deve essere limitato alla metà del coprioggetti. La ripida pendenza assicura il liquido scorre lungo la superficie del coprioggetti verso il basso, offrendo anche un sottile strato di poli-HEMA. Lasciare che il vetrini rivestiti di poli-HEMA ad asciugare nel cappuccio per 30-60 minuti a temperatura ambiente. </li><li> Segna il poli-HEMA lato del coprioggetti con una penna a punta fine vicino al bordo. </li><li> Praticare un foro di 1 cm nel fondo di un piatto della cultura 35 mm. </li><li> Incollare il poli-HEMA coprioggetti rivestiti al fondo del piatto con la cultura Sylgard 184 (Dow Corning Co., Midland, MI), in modo che la poli-HEMA lato patinato rivolto verso l&#39;interno del piatto. Assicurarsi che il bordo della poli-HEMA rivestimento corre lungo il centro del foro. Lasciare i piatti ad asciugare per 1-3 giorni a temperatura ambiente in un ambiente privo di polvere. </li><li> Scratch una linea sulla parte esterna del coprioggetti con un diamante punta della penna seguendo il bordo della poli-HEMA rivestimento. Poi, gratta due linee si intersecano ad angolo retto rispetto ad esso di circa due millimetri di distanza. I due punti di intersezione servire come riferimento o punti fiduciali da utilizzare per riprodurre lo stesso orientamento del piatto cultura sul palco microscopio. </li><li> Sterilizzare i piatti sotto la luce ultravioletta durante la notte. </li><li> Per creare una cella-aderente metà del piatto cultura, il cappotto non poli-HEMA metà con Sal-1, che contiene una salamandra anticorpi anti-topo sollevate contro le membrane delle cellule della retina (generosamente forniti da Dr. Peter MacLeish, Scuola di Medicina di Morehouse , Atlanta, GA; vedere MacLeish et al (1983)).. In primo luogo, il cappotto-1 Sal lato con circa 100μl di 0,1 mg / ml sterile capra anti-topo IgG (Boehringer Mannheim Corporation, Indianapolis IN), facendo attenzione a non versare il liquido sopra sulla poli-HEMA lato. Lasciate per tre ore, poi rimuoverlo. Lavare l&#39;area stessa con soluzione di Ringer sterile di una o due volte, poi posto 100μl Sal-1 surnatante, facendo attenzione a non versare il liquido nella poli-HEMA lato del piatto. Il primo anticorpo assicura Sal-1 è rivestita in una concentrazione nota. </li><li> Incubare i piatti con la Sal-1 durante la notte. </li><li> Rimuovere la soluzione Sal-1 anticorpo e sciacquare la zona con soluzione di Ringer sterile è. Aggiungere 2 ml di mezzo per il piatto appena prima di placcatura cellulare. </li></ol> La composizione della soluzione Ringer anfibi, mezzo privo di siero anfibi, e la soluzione di Ringer per la digestione enzimatica si trovano in MacLeish e Townes-Anderson (1988) e Mandell et al. (1993). Preparazione di colture cellulari Le cellule utilizzate in questo video sono stati ricavati dalla luce adattati, adulto, acquatico fase tigre salamandre (Ambystoma tigrinum), che misura 17-22 cm di lunghezza (Charles Sullivan Inc., Nashville, TN), mantenuto a 5 ° C su un 12 h: 12 h ciclo luce-buio. I protocolli sono stati approvati dalla cura degli animali e utilizzo Comitato Istituzionale (IACUC) presso l&#39;Università di Medicina e Odontoiatria del New Jersey in stretta conformità con le linee guida del National Institutes of Health. Le procedure per la preparazione di colture di cellule della retina sono descritti da Nachman-Clewner e Townes-Anderson (1996) e sono i seguenti: <ol><li> Decapitare midollo e gli animali. Rimuovere gli occhi e sezionare le cornee e iris. La retina deve staccare un po &#39;dentro l&#39;oculare. Inserendo una pinza curva sotto la retina, scoop le retine. Non è importante se l&#39;obiettivo è collegato alla retina in questa fase. </li><li> Digerire la retina in 14 U / ml di papaina (Worthington, Freehold, New Jersey) in soluzione di Ringer salamandra per 40 minuti. </li><li> Prima dell&#39;uso, la soluzione Ringer papaina dovrebbe essere bollito con% O 95 2 / 5% gas CO 2 per 30 min. E poi sterilizzati con passaggio attraverso un filtro di 0,22 micron (Millipore, Carrigtwohill, Irlanda). </li><li> Sciacquare la retina con una soluzione sterile di Ringer salamandra tre volte. </li><li> Togliere le lenti dalla soluzione del Ringer, poi delicatamente triturare il tessuto retinico con un foro da 3 mm pipetta fino ad ottenere una sospensione cellulare. </li></ol> Pinzette laser manipolazione dei neuroni isolati <ol><li> Porre la capsula cultura sul palco microscopio, orientando il piatto, allineando un segno sul piatto con un punto di riferimento sul palco. Salva le coordinate dei punti fiduciali nel computer. </li><li> Cellule piastra su entrambi i Sal-1 e poli-HEMA metà del piatto. </li><li> Consentire alle cellule di stabilirsi per circa 20 minuti, il che è abbastanza lungo per le cellule dalla parte aderente del piatto di allegare al substrato anticorpi. </li><li> Selezionare una cella sul lato Sal-1 aderenti del piatto. Segna il x e y coordinate fase di una posizione vicina alla cella. Nel nostro caso, il punto è stato di circa 10μm dalla dendriti primarie della cella selezionata. Salva coordinate nel computer. </li><li> Selezionare un neurone secondo, nel nostro caso un fotorecettore, sulla poli-HEMA lato del piatto e utilizzare lo strumento pinzetta laser per intrappolare esso. Cattura può essere raggiunto su una vasta gamma di livelli di potenza. Tuttavia, impostare la potenza del laser al 10% -20%, che è sufficientemente basso per evitare i detriti cattura insieme con il cellulare, ma sufficiente per trasportare il cellulare attraverso il mezzo. </li><li> Mentre si tiene la cellula nella trappola, abbassare il palco in modo che la cellula è ben al di sopra della superficie del piatto di sopra di qualsiasi cultura e neuroni attaccati. Spostare lo stadio sotto il controllo del computer per portare la cella a salvata in precedenza coordinate xey sul lato-1 Sal. Il movimento era pronto a 8-20μm / s, ma la velocità massima dovrebbe essere empiricamente determinato. All&#39;arrivo al punto di destinazione sul lato Sal-1, sollevare sul palco per portare la cellula intrappolati alla superficie del piatto. Posizionamento delle cellule può essere fatto con una precisione di pochi micron. Permettono alla cellula di aderire al substrato-1 Sal. </li><li> Ottenere immagini digitalizzate di entrambe le cellule nella coppia prima e dopo la formazione delle coppie fornisce un record utile. </li><li> Mantenere le coppie di cellule in un incubatore. Per le celle salamandra, posizionare i piatti in una camera umidificata a 10 ° C. Le cellule possono essere monitorate da lasso video in tempo e utilizzato con qualsiasi tecnica cellula biologica richiesti. </li></ol>

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

La luce ha slancio, e quando un raggio di luce viene rifratta che passa attraverso una cella, è necessaria una forza per cambiare la direzione del momento. A causa della legge di conservazione del momento, una forza nella direzione opposta deve, a sua volta, reagisce di nuovo su una cella. Ashkin (1991) ha mostrato che la forza generata da un fascio laser focalizzato da una lente obiettivo microscopio si muoverà una cella verso il centro della messa a fuoco. Anche se un raggio laser genera solo un paio di piconewtons di forza, questa forz...

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

using laser tweezers for manipulating isolated neurons in vitro

In questo articolo e video, si descrive i protocolli utilizzati nel nostro laboratorio per studiare le preferenze di targeting dei processi di rigenerazione delle cellule adulte in vitro i neuroni della retina. Procedure per la preparazione di colture di cellule della retina iniziare con la digestione dissezione, e triturazione della retina, e terminano con la placcatura di isolate cellule della retina sui piatti realizzati appositamente per l&#39;utilizzo con pinzette laser. Questi piatti sono divisi in una metà adesivo cellula e mezzo repellente cellula. Il lato adesivo cella è rivestita con uno strato di Sal-1 anticorpi, che forniscono un substrato su cui le nostre cellule crescono. Altri substrati adesivi potrebbero essere utilizzati per altri tipi di cellule. Il lato repellente cellula è rivestita con un sottile strato di poli-HEMA. Le cellule placcato sulla poli-HEMA lato del piatto sono intrappolati con le pinzette laser, trasportate e poi messo in una cella adiacente sul lato Sal-1 per creare una coppia. Formazione di gruppi di cellule di qualsiasi dimensione dovrebbe essere possibile con questa tecnica. &quot;Laser-pinzette controllato micromanipolazione&quot; significa che il ricercatore può scegliere le celle da spostare, e la distanza desiderata tra le cellule possono essere standardizzati. Poiché il raggio laser passa attraverso superfici trasparenti del piatto cultura, la selezione delle cellule e il posizionamento sono fatte in ambiente chiuso ambiente sterile. Le cellule possono essere monitorati tramite video time-lapse e utilizzato con qualsiasi tecnica cellula biologica richiesti. Questa tecnica può aiutare indagini di interazioni cellula-cellula.

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

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

Questo video descrive la manipolazione di colture di neuroni utilizzando pinzette laser in vitro.

Utilizzando pinzette laser per manipolare i neuroni isolato in vitro

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

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

Research

JoVE Journal

Biology

10.3K Views.  University of Medicine and Dentistry of New Jersey - UMDNJ. Questo video descrive la manipolazione di colture di neuroni utilizzando pinzette laser in vitro.

Video: Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

Measuring Exocytosis in Neurons Using FM Labeling

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


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

Misurare esocitosi nei neuroni Utilizzando Etichettatura FM

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

Ricerca

Biologia

Silicon Microchips for Manipulating Cell-cell Interaction

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

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

I microchip di silicio per la manipolazione di interazione cellula-cellula

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

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

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

Utilizzando Micro-Electro-Mechanical Systems (MEMS) per sviluppare strumenti diagnostici

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

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

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

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

Analisi di Schwann-astrociti Interazioni utilizzo In Vitro Saggi

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

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

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

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

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

Isolamento dei ribosomi Polipeptidi Bound nascenti<em&gt; In vitro</em&gt; Per identificare i siti di pausa traslazione lungo mRNA

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

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

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

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

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

In Vitro aggregazione Assays Utilizzando iperfosforilata Tau Protein

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

Manipulating the Murine Lacrimal Gland

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

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

Manipolare il murino Ghiandola lacrimale

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

The Mouse Isolated Perfused Kidney Technique

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

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

La tecnica del rene perfusi mouse isolato

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

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

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

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

Analisi delle sospensioni cellulari isolate da tessuti solidi da Spectral citometria a flusso

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

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

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

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