La recherche a été financée par des subventions du NIH et de EY012031 EY0182175 et le FM Kirby Fondation.

Pinces optiques permettant de manipuler des cellules isolées en culture Les forces de piégeage des pinces optiques sont générées à partir de l&#39;élan de la lumière (Ashkin, 1991;. Ashkin et al, 1986). Bien que ces forces facilement piéger les cellules en suspension, ils ne sont pas en mesure de déplacer des cellules qui adhèrent à une surface. Par conséquent, pour réduire l&#39;adhérence de la boîte de culture à l&#39;endroit où les cellules sont piégées, la lamelle est enduit avec de la poly-2-hydroxyéthylméthacrylate (poly-HEMA) (Sigma Chemical Co., St Louis, MO), un composé non toxique aux cellules répulsif propriétés précédemment employé pour les études de l&#39;adhérence sur les cellules endothéliales (Folkman et Moscona, 1978). Alors que le poly-HEMA couvre la moitié de la boîte de culture, l&#39;autre moitié est recouvert d&#39;un substrat qui supporte la croissance cellulaire. Dans notre laboratoire, Sal-1 est utilisé comme substrat pour les cellules de la rétine de salamandre culture. La procédure pour la fabrication de Sal-1 et poly-HEMA plats enduit est décrit ci-dessous: Fabrication de boîtes de culture <ol><li> Coverglasses propre (# 1 VWR Scientific Inc, Media, PA) dans l&#39;acide. Cette lamelle a été sélectionné pour son épaisseur de seulement 0.1mm, nécessaires pour atteindre les objectifs de haute ouverture numérique nécessaire pour la focalisation du laser. </li><li> Pour créer une zone de cellules répulsif, dissoudre 20 mg de poly-HEMA (Sigma Chemical Co., St Louis, MO) dans 1 ml d&#39;éthanol à 95% pendant une nuit à température ambiante. De préférence, cette solution doit être utilisée dans un mois de préparation. </li><li> Caler l&#39;coverglasses à une position presque verticale dans un environnement exempt de poussière, comme un capuchon. </li><li> Placez une ou deux gouttes de solution de poly-HEMA près du sommet de la lamelle de la pointe en plastique d&#39;une micropipette 200 pl. Les gouttes (environ 50 100 pi-chacun) devrait être limité à la moitié de la lamelle. La pente raide assure que le liquide coule de la surface du couvre-objet au fond, fournissant un enduit mince voire de poly-HEMA. Laisser le coverglasses poly-HEMA revêtement à sécher dans la hotte pendant 30-60 minutes à température ambiante. </li><li> Marquer le côté poly-HEMA de l&#39;lamelle avec un stylo à pointe fine près du bord. </li><li> Percez un trou de 1 cm dans le fond d&#39;une boîte de culture de 35 mm. </li><li> Collez la lamelle poly-HEMA revêtement au fond de la boîte de culture avec des Sylgard 184 (Dow Corning Co., Midland, MI) afin que le côté poly-HEMA recouvert visages vers l&#39;intérieur du plat. S&#39;assurer que le bord de la couche de poly-HEMA parcourt le centre du trou. Autoriser les plats à sécher pendant 1-3 jours à température ambiante dans un environnement exempt de poussière. </li><li> Scratch une ligne sur l&#39;extérieur de la lamelle avec un stylo à pointe de diamant en suivant le bord du revêtement en poly-HEMA. Ensuite, gratter deux lignes se croisent à angle droit de la part d&#39;environ 2mm. Les deux points d&#39;intersection servir de référence ou des points de repères à utiliser pour reproduire la même orientation de l&#39;antenne de la culture sur la platine du microscope. </li><li> Stériliser les plats sous la lumière ultraviolette pendant la nuit. </li><li> Pour créer une demi-pile-adepte de la boîte de culture, le manteau de la non poly-HEMA moitié avec Sal-1, qui contient une souris anti-salamandre anticorps dirigé contre les membranes des cellules rétiniennes (généreusement fourni par le Dr Peter MacLeish, Morehouse School of Medicine , Atlanta, GA, voir MacLeish et al (1983)).. Premièrement, le manteau de la Sal-1 à côte avec environ 100 pi de 0,1 mg / ml stériles de chèvre anti-IgG de souris (Boehringer Mannheim Corporation, Indianapolis IN), en prenant soin de ne pas renverser le liquide sur sur le côté poly-HEMA. Laisser pendant trois heures, puis l&#39;enlever. Laver la même zone avec une solution Ringer stérile une fois ou deux, puis placez 100 ul Sal-1 surnageant, en prenant soin de ne pas renverser le liquide dans le côté poly-HEMA du plat. Le premier anticorps assure Sal-1 est enduit à une concentration connue. </li><li> Incuber les plats avec la nuit Sal-1. </li><li> Retirer la solution Sal-1 anticorps et rincer la zone avec une solution Ringer stérile. Introduisez 2 ml de milieu à l&#39;antenne juste avant le placage cellule. </li></ol> La composition de la solution de Ringer amphibien, un milieu sans sérum d&#39;amphibiens, et la solution de Ringer pour la digestion enzymatique sont trouvés dans MacLeish et Townes-Anderson (1988) et Mandell et al. (1993). Préparation des cultures de cellules Les cellules utilisées dans cette vidéo ont été tirées de la lumière adaptée, des adultes, aquatiques phases de tigre salamandres (Ambystoma tigrinum), mesurant de 17 à 22 cm de longueur (Charles Sullivan Inc, Nashville, TN), maintenu à 5 ° C sur un 12 h: 12 h cycle lumière-obscurité. Les protocoles ont été approuvés par l&#39;Institutional Animal Care et utilisation comité (IACUC) à l&#39;Université de médecine et de dentisterie du New Jersey en stricte conformité avec les directives du National Institutes of Health. Les procédures de préparation des cultures de cellules rétiniennes sont décrits par Nachman-Clewner et Townes-Anderson (1996) et sont comme suit: <ol><li> Décapiter et la moelle des animaux. Retirez les yeux et disséquer les cornées et d&#39;iris. Les rétines doit se détacher légèrement dans l&#39;œilleton. En plaçant une pince courbe sous la rétine, évider les rétines. Il n&#39;est pas important si la lentille est attaché à la rétine à ce stade. </li><li> Digest de la rétine dans 14 U / ml papaïne (Worthington, Freehold, New Jersey) dans une solution de Ringer salamandre pendant 40 minutes. </li><li> Avant l&#39;utilisation, la solution de Ringer papaïne devrait faire barboter avec 95% O 2 / 5% de CO 2 de gaz pendant 30 min., Puis stérilisés par passage à travers un filtre de 0,22 micron (Millipore, Carrigtwohill, Irlande). </li><li> Rincez les rétines avec une solution stérile de la salamandre de Ringer à trois reprises. </li><li> Retirer les lentilles de la solution du Ringer, puis doucement triturer les tissus rétiniens avec un alésage de 3 mm pipette pour obtenir une suspension cellulaire. </li></ol> Laser pinces de manipulation de neurones isolés <ol><li> Placer le plat de culture sur la platine du microscope, en orientant le plat en alignant une marque sur le plat avec un point de référence sur la scène. Enregistrer les coordonnées des points de repères dans l&#39;ordinateur. </li><li> Cellules de la plaque sur les deux Sal-1 et poly-HEMA moitiés du plat. </li><li> Permettre aux cellules de se contenter d&#39;environ 20 minutes, ce qui est assez long pour les cellules adhérentes sur le côté du plat à joindre au substrat d&#39;anticorps. </li><li> Sélectionnez une cellule sur le côté de Sal-1 adhérente du plat. Marquer le x et le stade y les coordonnées d&#39;une position proche de la cellule. Dans notre cas, le point a été d&#39;environ 10 microns des dendrites primaires de la cellule sélectionnée. Enregistrer les coordonnées dans l&#39;ordinateur. </li><li> Sélectionnez un deuxième neurone, dans notre cas un photorécepteur, sur le côté poly-HEMA du plat et utilisez l&#39;outil pince au laser pour le piéger. Le piégeage peut être réalisé sur une large gamme de niveaux de puissance. Toutefois, nous avons mis la puissance du laser à 10% -20%, ce qui est assez bas pour éviter les débris piégeage avec la cellule, mais suffisante pour le transport de la cellule par l&#39;intermédiaire. </li><li> Tout en maintenant la cellule dans le piège, baissez le stade de sorte que la cellule est bien au-dessus de la surface de la boîte de culture et surtout toute neurones attachés. Déplacer le stade sous contrôle informatique pour apporter à la cellule de l&#39;précédemment enregistré coordonnées x et y sur la Sal-1 côté. Le mouvement scénique a été fixé à 8-20μm / s, mais la vitesse maximale doit être déterminée empiriquement. A l&#39;arrivée au point de destination sur le Sal-1 côté, soulevez le stade pour apporter la cellule piégée à la surface du plat. Le placement de cellules peuvent être faites avec une précision de quelques microns. Permettre à la cellule d&#39;adhérer à la Sal-1 substrat. </li><li> Obtenir des images numérisées de deux cellules de la paire avant et après la formation de paires fournit un compte rendu utile. </li><li> Maintenir les paires de cellules dans un incubateur. Pour les cellules de salamandre, placer la vaisselle dans une chambre humidifiée à 10 ° C. Les cellules peuvent être surveillés par laps de temps la vidéo et utilisés avec n&#39;importe quelle technique de biologie cellulaire nécessaires. </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 lumière a l&#39;élan, et quand un rayon de lumière est réfractée lorsqu&#39;elle passe à travers une cellule, une force est nécessaire pour changer la direction de l&#39;élan. En raison de la loi de conservation de l&#39;élan, une force dans la direction opposée doivent, à leur tour, réagissent à leur tour sur une cellule. Ashkin (1991) ont montré que la force générée par un faisceau laser focalisé par un objectif de microscope se déplace d&#39;une cellule vers le centre d&#39;intérêt. Même si un faisceau laser ne g...

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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

Dans ce papier et vidéo, nous décrivons les protocoles utilisés dans notre laboratoire pour étudier les préférences de ciblage des processus régénérant cellulaire des neurones rétiniens adultes in vitro. Procédures pour préparer des cultures de cellules rétiniennes commencer par la dissection, la digestion et la trituration de la rétine, et se terminent avec le placage de cellules rétiniennes isolées sur des plats spécialement conçus pour une utilisation avec pinces optiques. Ces plats sont divisés en une demi adhésion cellulaire et demi répulsif cellule. Le côté adhésif cellule est recouverte d&#39;une couche de Sal-1 anticorps, qui fournissent un substrat sur lequel les cellules de notre croissance. D&#39;autres substrats adhésif pourrait être utilisé pour d&#39;autres types cellulaires. Le côté répulsif cellule est recouverte d&#39;une fine couche de poly-HEMA. Les cellules étalées sur le côté poly-HEMA du plat sont piégés avec la pince à épiler au laser, transporté et placé à proximité d&#39;un cellulaire sur la Sal-1 côté pour créer une paire. Formation des groupes de cellules de n&#39;importe quelle taille devrait être possible avec cette technique. «Laser-pincette contrôlée micromanipulation» signifie que le chercheur peut choisir lequel les cellules à se déplacer, et la distance souhaitée entre les cellules peuvent être standardisés. Parce que le faisceau laser passe à travers des surfaces transparentes de la boîte de culture, la sélection de cellules et de placement sont effectués dans un espace clos, environnement stérile. Les cellules peuvent être surveillés par vidéo time-lapse et utilisé avec n&#39;importe quelle technique de biologie cellulaire nécessaires. Cette technique peut aider les enquêtes sur les interactions cellule-cellule.

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

Cette vidéo décrit la manipulation des neurones cultivés en utilisant le laser pincettes in vitro.

L&#39;aide de pincettes laser pour la manipulation neurones isolés 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 vues.  University of Medicine and Dentistry of New Jersey - UMDNJ. Cette vidéo décrit la manipulation des neurones cultivés en utilisant le laser pincettes in vitro.

Vidéo: 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.

Mesure exocytose dans les neurones en utilisant un étiquetage 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 ...

Recherche

Biologie

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.

Les micropuces Silicon pour la manipulation d&#39;interaction cellule-cellule

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

Utiliser Micro-Electro-Mechanical Systems (MEMS) pour développer des outils diagnostiques

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.

Analyse de Schwann-astrocytes interactions à l&#39;aide In Vitro Tests

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.

Isolement du ribosome Bound polypeptides naissants<em&gt; In vitro</em&gt; Pour identifier les sites de pause de translation le long d&#39;ARNm

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 agrégation essais utilisant la protéine tau hyperphosphorylée

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.

Manipuler le murin Glande lacrymale

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 perfusé Technique du rein de souris isolé

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.

Analyse des suspensions de cellules isolées de tissus solides par Spectral cytométrie en flux

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.