Solutions <ul><li> Solution intracellulaire des cellules ganglionnaires (en mm): 100 K-gluconate, 8 KCl, MgCl 1 2, 1 EGTA, HEPES 10, 4 ATP, GTP 0,5, 90 uM Sulforhodamine B (pour la coloration), ajusté à pH = 7,4 avec KOH </li><li> Solution intracellulaire des cellules bipolaires (en mm): 90 K-gluconate, 8 KCl, MgCl 2 1, 10 BAPTA, HEPES 10, 4 ATP, GTP 0,5, 90 uM Sulforhodamine B (pour la coloration), ajusté à pH = 7,4 avec KOH </li><li> Ringer extracellulaire de solution (en mM): 112 NaCl, glucose à 5, 5 HEPES, 2 KCl, CaCl 2 2, 1 MgCl2, ajusté à pH = 7,75 avec NaOH. Oxygénation de la solution n&#39;est pas nécessaire. </li></ul> Préparer chambres d&#39;enregistrement Notre chambre d&#39;enregistrement se compose d&#39;une lame de microscope avec une colle bien à ce qui bloque en silicone petits découpées dans une autre colle bien placés pour assurer la stabilité des tranches de tissu. Préparation des tranches de la rétine Pour éviter le blanchiment des photorécepteurs, la dissection est menée à la lumière de lumière IR ou rouge faible. Si l&#39;on s&#39;intéresse à l&#39;enregistrement des réponses de la voie de la tige, que la lumière infrarouge doit être utilisé (IR loupe binoculaire et aucune lumière ambiante. Supplémentaire des lunettes IR devrait être utilisé pour des procédures ne se fait pas sous le microscope). Si on s&#39;intéresse principalement à l&#39;enregistrement des réponses de lumière conique, faible lumière ambiante rouge ou un feu rouge clignotant peut être utilisé. Le microscope, cependant, devrait encore avoir oculaires IR. Si l&#39;entrée photorécepteur n&#39;est pas pertinent pour l&#39;expérience, les procédures peuvent être effectuées à la lumière ambiante normale en utilisant un microscope ordinaire. <ol><li> Préparer deux lames de microscope avec une colle silicone ainsi: créer deux bandes parallèles de la graisse de silicone (environ 0,5 cm de distance) sur chaque lame et placer un morceau de papier rectangulaire de filtre sur un côté de la diapositive, en traversant les deux bandes de graisse (pousser le filtre papier avec le cadre d&#39;une lame de rasoir, de sorte qu&#39;il est bien attaché à la lame) </li><li> Sacrifice une salamandre selon des protocoles établis: l&#39;animal est placé dans un bain de glace pendant 30 minutes, puis décapité et double-décérébrés. </li><li> Supprimer les yeux de la tête salamandre et le placer sous le microscope de dissection sur un morceau de papier de soie humide avec une solution glacée de Ringer </li><li> Retirez le tissu conjonctif de l&#39;oeil </li><li> Utilisez une lame de rasoir pour faire une petite incision dans la cornée </li><li> Tenez coupé la cornée avec une pince lors de son retrait avec des ciseaux le long de sa pièce jointe à l&#39;oeil </li><li> Retirez la lentille avec une pince </li><li> Tenir l&#39;iris avec une pince et couper le long de l&#39;ora serrata pour enlever l&#39;iris </li><li> Couper l&#39;œilleton en deux morceaux rectangulaires </li><li> Ramassez l&#39;une des pièces latérales et placez-le jusqu&#39;à la sclère sur le papier filtre (veillez à s&#39;aplatir la pièce tout en la poussant vers le bas avec beaucoup d&#39;attention) </li><li> Soulevez doucement la sclère sur la rétine, qui désormais doit être jointe au document de filer </li><li> Ajouter rapidement solution glacée de Ringer au bien </li><li> Répétez la même chose pour le deuxième morceau </li><li> Utilisez une lame de rasoir placée dans une trancheuse pour couper sur mesure de 200 300 um tranches de large. </li><li> Utilisez une paire de pinces pour ramasser les tranches sur le côté du papier filtre, les retourner afin couches de la rétine deviennent visibles, et les placer sur les bandes de graisse à vide </li><li> Choisissez une tranche qui ressemble en bon état et présente des bandes horizontales indiquant les couches de la rétine (les couches sont parfois difficiles à voir sous le microscope de dissection que le grossissement n&#39;est pas assez élevé, cependant, si l&#39;on peut reconnaître une certaine superposition de la tranche est généralement assez bon. La décision finale quant à l&#39;utilisation de la tranche, cependant, est faite une fois qu&#39;il est considéré sous le microscope vertical). C&#39;est passer à la diapositive chambre d&#39;enregistrement en construisant un pont Ringer entre les deux lames </li><li> Assurez-vous que le papier filtre est poussé contre les blocs de caoutchouc dans la chambre d&#39;enregistrement de sorte que la tranche n&#39;est pas incliné (cellules ganglionnaires et des photorécepteurs doit être mise au point dans le même temps) </li></ol> Placer le tissu dans la plate-forme d&#39;enregistrement <ol><li> La diapositive d&#39;enregistrement est placé sous le microscope et perfusées avec des solutions de Ringer à travers une perfusion par gravité systé </li><li> La préparation est visualisé avec un microscope debout avec un objectif à immersion d&#39;eau 10x et 40x. </li><li> Encore une fois, nous utilisons uniquement la lumière infrarouge pour empêcher le blanchiment des photorécepteurs. Une caméra infrarouge est attaché au port côté du microscope, qui est relié à un écran de télévision. </li></ol> Stimulation visuelle Les stimuli visuels sont programmés en Matlab utilisant la Psychtoolbox et sont projetées sur la rétine grâce à l&#39;orifice supérieur du microscope en utilisant une image de microscopie injecteur (MBF biosciences). Un Bits + Processeur + (Digital Video Cambridge Research Systems) est utilisé pour obtenir une échelle de luminance 14bit et synchronize présentation du stimulus avec acquisition de données. Les stimuli visuels utilisés dans cette vidéo des bars lumineux ou sombres (largeur = 460μm) de contraste de 100%, qui ont été présentés pendant deux secondes sur un fond uniforme régulier (luminance = 8 * 10 4 photons / um / s, taille: 1,84 x 1.38mm). Électrophysiologie <ul><li> Patch électrodes sont tirés de verre borosilicate (OD:.. 1.5mm, ID 84mm) avec un extracteur micropipette. Pour les cellules ganglionnaires, les résistances sont entre 2 et 4 MOhms. Pour les cellules bipolaires, plus les résistances entre 4 et 8 MOhms sont utilisés. </li><li> Enregistrements voltage sont effectuées en utilisant un 700A Multiclamp (Molecular Devices), un amplificateur et un laboratoire logiciel interne programmé dans LabVIEW. </li><li> Les médicaments sont perfusés à travers un système de perfusion à 8 canaux. L&#39;électrode microperfusion est placée près de la tranche (le tissu doit visiblement se déplacer quand vous tournez la perfusion et l&#39;éteindre, mais il faut faire attention à ce que la pression n&#39;est pas trop fort pour que la tranche pourrait se détacher du papier filtre). Au cours des expériences de la microperfusion doit être constamment allumé (solution de contrôle si aucune perfusant médicaments sont nécessaires) afin que l&#39;enregistrement ne soit pas perturbé par la perfusion de commutation sur et en dehors. </li></ul>

<ol>
	<li>Cook, P. B., Lukasiewicz, P. D., McReynnolds, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA(C)+receptors+control+adaptive+changes+in+a+glycinergic+inhibitory+pathway+in+salamander+retina.">GABA(C) receptors control adaptive changes in a glycinergic inhibitory pathway in salamander retina.</a> Journal of Neuroscience. 20, 806-812 (2000).</li><li>Heflin, S. J., Cook, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Narrow+and+wide+field+amacrine+cells+fire+action+potentials+in+response+to+depolarization+and+light+stimulation.">Narrow and wide field amacrine cells fire action potentials in response to depolarization and light stimulation.</a> Visual Neuroscience. 24, 197-206 (2007).</li><li>Ichinose, T., Shields, C. R., Lukasiewicz, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+Channels+in+the+Transient+Retinal+Bipolar+Cells+Enhance+Visual+Responses+in+Ganglion+Cells.">Sodium Channels in the Transient Retinal Bipolar Cells Enhance Visual Responses in Ganglion Cells.</a> J Neurosci.. 25, 1856-1865 (2005).</li></ol>

Avantages: <ol><li> Tous les types de cellules sont accessibles </li><li> Identification aisée des différents types cellulaires, en particulier si un colorant fluorescent est ajouté à la solution d&#39;électrode </li><li> Les agents pharmacologiques peuvent facilement atteindre les cellules cibles </li><li> La technique de patch-clamp permet d&#39;étudier le rôle des canaux ioniques différentes dans les calculs de la rétine. La même information ne peut être obtenu grâce à des enregistrements ou des enregis...

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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>Multiclamp 700A</td>
<td>Patch Clamp Amplifier</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>SZX9</td>
<td>Dissection Micropscope</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BX50WI</td>
<td>Upright Microscope</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Equipped with 40x, 10x water immersion objective, fluorescent filters and mercury lamp</td>
</tr>
<tr>
<td>P-97</td>
<td>Micropipette Puller</td>
<td>Sutter Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Lucivid</td>
<td>Microscope image injector</td>
<td>Mbf bioscience</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>8-channel perfusion system</td>
<td>Microperfusion</td>
<td>Parker Hannfin corporation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bits++</td>
<td>Digital Video Processor</td>
<td>Cambridge Research Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Infrared Oculars</td>
<td>Other</td>
<td>ITT</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Adhesive silicone wells</td>
<td>Other</td>
<td>Molecular Probes</td>
<td>&nbsp;</td>
<td>20mm diameter, 1.0mm deep</td>
</tr>
<tr>
<td>Membrane Filters</td>
<td>Filter</td>
<td>Milipore</td>
<td>HAWPO1300</td>
<td>.45&#181;m HA</td>
</tr>
<tr>
<td>Borosilicate Glass Capillaries</td>
<td>Electrode glass</td>
<td>World Precision Instruments</td>
<td>1B150-4</td>
<td>&nbsp;</td>
<tr>
<td>Syringe Filters</td>
<td>Filter</td>
<td>Whatman</td>
<td>6789-0402</td>
<td>4mm filters, .2um Nylon Membrane, Polypropylene housing</td>
</tr>
<tr>
<td>IR camera</td>
<td>Micropscope mounted camera</td>
<td>Sony</td>
<td>SPT-M324</td>
<td>Extrawave HAD, B7W video camera</td>
</tr>
<tr>
<td>Picrotoxin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P1675</td>
<td>&nbsp;</td>
<tr>
<td>Strychnine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S0532</td>
<td>&nbsp;</td>
<tr>
<td>Imidazole-4-acetic acid sodium salt</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I7013</td>
<td>&nbsp;</td>
<tr>
<td>Larval tiger salamanders</td>
<td>Animal</td>
<td>Charles E. Sullivan (Nashville, TN)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

whole-cell recordings of light evoked excitatory synaptic currents in the retinal slice

Nous utilisons la technique de cellules entières patch clamp pour étudier les circuits synaptiques qui sous-tend le traitement des informations visuelles de la rétine. Dans cette vidéo, nous allons vous guider à travers le processus d&#39;exécution de cellules entières d&#39;enregistrements de la lumière des courants évoqués des cellules individuelles dans la préparation de tranches de la rétine. Nous utilisons la salamandre tigrée aquatiques comme un modèle animal. Nous commençons par décrire la dissection de l&#39;oeil et de montrer comment les tranches sont montés pour des enregistrements électrophysiologiques. Une fois que la tranche est placée dans la chambre d&#39;enregistrement, nous montrent comment effectuer la cellule entière enregistrements voltage. Nous avons ensuite projet de stimuli visuels sur les photorécepteurs de la tranche à susciter la lumière évoque les réponses actuelles. Pendant l&#39;enregistrement, nous perfuser la tranche avec des agents pharmacologiques, par lequel un système de perfusion à 8 canaux nous permet de basculer rapidement entre les différents agents. La préparation de tranches rétiniennes est largement utilisé pour les enregistrements de patch-clamp dans la rétine, en particulier d&#39;étudier ou de cellules amacrines bipolaire, qui ne sont pas accessibles dans une préparation toute monture.

Watch this Scientific Journal Video about Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice at JoVE.com

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

Cette vidéo montre le processus de la cellule entière enregistrements pince de tension dans la tranche la rétine de la salamandre aquatique tigre. Nous démontrons la préparation de la tranche ainsi que la façon d&#39;effectuer des enregistrements de patch-clamp pendant la stimulation visuelle de la rétine.

Cellules entières Recordings of Light évoqués courants synaptiques excitateurs dans la tranche la rétine

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

whole-cell-recordings-light-evoked-excitatory-synaptic-currents

Research

JoVE Journal

Biology

12.7K vues.  Boston University. Cette vidéo montre le processus de la cellule entière enregistrements pince de tension dans la tranche la rétine de la salamandre aquatique tigre. Nous démontrons la préparation de la tranche ainsi que la façon d'effectuer des enregistrements de patch-clamp pendant la stimulation visuelle de la rétine.

Vidéo: Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

Horizontal Slice Preparation of the Retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of retinal neurons, such as amacrine cells, expand their dendrites horizontally, so that the morphology of the cells is supposed to be severely damaged in the vertical slices. In the whole-mount preparation, especially for patch-clamp recordings, retinal neurons in the middle layer are not easily accessible due to the extensive coverage of glial cell (Mueller cell) s endfeets. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. The slice was made horizontally at the inner layer of the retina using a vibratome slicer after the retina was embedded in the low-temperature melting agarose gel. In this horizontal slice preparation of the retina, we studied the function of retinal neurons compared with their morphology, by using patch-clamp recording, calcium imaging technique, immunocytochemistry, and single-cell RT-PCR.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.

Préparation tranche horizontale de la rétine

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

Recherche

Biologie

Whole Cell Recordings from Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown.  The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.

This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.

Enregistrements à partir de cellules entières du cerveau d&#39;adulte drosophile

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

Principales composantes d&#39;un microscope optique

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Recordings of Neural Circuit Activation in Freely Behaving Animals

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. Traditional non-invasive methods require at least partially restrained research subjects, and they only allow identification of large numbers of simultaneously activated neurons. On the other hand, small ensembles of neurons or individual neurons can only be measured using single-cell recordings obtained from largely reduced preparations. Since the expression of natural behavior is limited in restrained and dissected animals, the underlying neural mechanisms that control such behavior are difficult to identify. 
Here, I present a non-invasive physiological technique that allows measuring neural circuit activation in freely behaving animals. Using a pair of wire electrodes inside a water-filled chamber, the bath electrodes record neural and muscular field potentials generated by juvenile crayfish during natural or experimentally evoked escape responses. The primary escape responses of crayfish are mediated by three different types of tail-flips which move the animals away from the point of stimulation. Each type of tail-flip is controlled by its own neural circuit; the two fastest and most powerful escape responses require activation of different sets of large &#8220;command&#8221; neurons. In combination with behavioral observations, the bath electrode recordings allow unambiguous identification of these neurons and the associated neural circuits. Thus activity of neural circuitry underlying naturally occurring behavior can be measured in unrestrained animals and in different behavioral contexts.

Non-invasive measurements of neural activity patterns in freely behaving animals are obtained by combining neurophysiological recordings with high speed videography.

Enregistrements de l&#39;activation des circuits neuronaux dans les Librement Behaving Animaux

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Enregistrements aspiration simple cellule à partir de cônes photorécepteurs de la souris

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated Calcium (CRAC) channels. CRAC channels play important role in regulation of T cell proliferation and gene expression. Abnormal CRAC channel function in T cells has been linked to severe combined immunodeficiency and autoimmune diseases 1, 2 . Studying CRAC channel function in human T cells may uncover new molecular mechanisms regulating normal immune responses and unravel the causes of related human diseases. Electrophysiological recordings of membrane currents provide the most accurate assessment of functional channel properties and their regulation. Electrophysiological assessment of CRAC channel currents in Jurkat T cells, a human leukemia T cell line, was first performed more than 20 years ago 3, however, CRAC current measurements in normal human T cells remains a challenging task. The difficulties in recording CRAC channel currents in normal T cells are compounded by the fact that blood-derived T lymphocytes are much smaller in size than Jurkat T cells and, therefore, the endogenous whole-cell CRAC currents are very low in amplitude. Here, we give a step-by-step procedure that we routinely use to record the Ca2+ or Na+ currents via CRAC channels in resting human T cells isolated from the peripheral blood of healthy volunteers. The method described here was adopted from the procedures used for recording the CRAC currents in Jurkat T cells and activated human T cells 4-8.

Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

We provide a step-by-step protocol for whole-cell patch clamp recording of Calcium Release-Activated Calcium (CRAC) currents in peripheral blood mononuclear cell-derived human T lymphocytes.

Cellules entières d&#39;enregistrement de Calcium Release-Activated calcium (CRAC) Courants dans les lymphocytes T humains

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

L&#39;étude de la protéolyse de la cycline B au niveau de la cellule unique dans les populations de cellules entières

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

La méthode la Culture Slice pour la suite du développement des germes de dents dans explantation Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo suivi 4-Dimensional de hématopoïétiques cellules souches et progénitrices adultes dans la voûte du crâne de souris de moelle osseuse

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Méthode d&#39;évaluation des effets d&#39;une gamme de longueurs d&#39;onde et des intensités de Luminothérapie Rouge / proche infrarouge sur le stress oxydatif<em&gt; In Vitro</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...