NZ tiens à remercier les membres du laboratoire Subhas Biswas, Andrew Sproul, et Ryan Willet pour sa formation en disséquant et la récolte neurones sympathiques. Soutenu par des subventions du NIH-NINDS.

1. Préparation à la dissection: <ol><li> Sélectionnez la boîte de culture approprié (10 cm ou des plaques 6 puits ou 24 puits, etc) selon la nature de vos expériences, et les enduire de collagène de queue de rat 24 heures avant la dissection. Les méthodes de préparation de collagène de queue de rat et l&#39;utiliser pour des boîtes de culture manteau ont été décrites ailleurs (1). </li><li> Préparer une solution de trypsine à 0,25% en tampon phosphate salin (PBS) (sans EDTA) et stériliser par filtration. Préparer des aliquotes de 1 ml et conserver à -20 ° C. </li><li> Préparer une solution contenant 2,5 mM de chacun des uridine et de 5-FDU dans l&#39;eau distillée / désionisée H 2 O. Filtre-stériliser et préparer 50 aliquotes. Entreposer à -20 º C. </li><li> Préparer le milieu complet: milieu de culture RPMI 1640 avec inactivés par la chaleur de 10% de sérum de cheval (chaleur inactiver en incubant dans un bain-marie 56 º C pendant 30 minutes) et 5% de sérum fœtal bovin. </li><li> Préparer milieu final: milieu de culture RPMI 1640 avec inactivés par la chaleur de 1% de sérum de cheval + NGF (100ηg/ml concentration finale) + pénicilline / streptomycine (1 / 250) + 1 / 250 uridine/5-FDU solution (concentration de 10 uM final) . Remarque: ce moyen doit être constitué fraîches avant de les utiliser à chaque fois. Pour une expérience dans un format de plaque de 24 puits, on aura besoin pour préparer 12 ml de ce milieu. Il est recommandé de préparer un petit supplément. NGF peuvent être achetés auprès de diverses sources commerciales (voir le tableau à la fin de ce protocole). </li><li> Equipement: Un besoin d&#39;une loupe binoculaire et la source lumineuse. Un illuminateur col de cygne double fibre optique avec des conseils au point est préféré. En outre, on aura besoin pour nettoyer et stériliser (par autoclavage ou par trempage dans de l&#39;éthanol 70% pendant au moins 20 minutes) les outils de dissection suivante: deux paires de pinces à disséquer les micro-acier inoxydable, et une paire de ciseaux de dissection (également en acier inoxydable et de deux de Roboz). Deux aiguilles de seringues stériles (26 x gague 1 ½ pouces ou similaire) seront également nécessaires. Il est nécessaire de mettre en place un conseil de dissection, qui est un 3 &quot;x3&quot; morceau de styromousse enveloppé dans du papier aluminium et recouvert d&#39;une Kimwipe. Vaporiser le conseil d&#39;éthanol à 70% pour décontaminer. Enfin, préparer un feu poli pipette en verre pâturages. Afin de feu polonais, prendre la pipette en verre et maintenez la pointe de la flamme pendant quelques secondes, tout en tournant. Lors de l&#39;inspection, la pointe sera un aspect plus lisse et l&#39;ouverture sera plus étroite. Cela est nécessaire pour l&#39;étape où les cellules seront dissociées. Effectuer poli-feu dans une hotte de culture cellulaire de telle sorte que la pipette reste stérile. </li></ol> 2. La dissection de ganglions cervicaux Supérieur: <ol><li> Recueillir les ratons nouveau-nés qui sont en âge de P0 ou P1. </li><li> Rapidement essuyez le chiot pour la dissection à l&#39;éthanol 70% pour réduire les risques de contamination. </li><li> Rapidement décapiter chiot avec une paire de ciseaux (cette étape ne sera pas montré dans la vidéo). Tenir la tête à l&#39;aide des pinces stériles de gaze stérile contre les égoutter brièvement excès de sang. </li><li> Placez la tête sur le pavé de dissection avec la face ventrale vers le haut et l&#39;extrémité caudale orientée vers vous. La trachée coupée doit être facilement visible. Utiliser les aiguilles de seringues stériles pour sécuriser la tête coupée au pad dissection de la manière suivante: pousser une broche si le toit de la bouche dans le pad et poussez la seconde broche si la trachée coupée dans le coussinet. Placez le coussin sous le microscope de dissection et de commencer la dissection. </li><li> En utilisant les deux pinces (une dans chaque main) la peau claire loin et de graisse autour de la trachée, en prenant soin de ne pas aller trop profond. Faire cela va exposer une paire de muscles courant presque parallèlement les uns aux autres de chaque côté de la trachée. Remarque: lors de la dissection, vous aurez besoin d&#39;irriguer avec du PBS stérile à froid ou à froid, sans sérum du milieu RPMI 1640 livré avec une pipette stérile, Pasteur pour laver l&#39;excès de sang et de garder l&#39;endroit propre et humide. Cela peut être fait qu&#39;une seule fois ou plusieurs fois selon la vitesse on travaille. Pour un dissecteur expérience, il ne prend que deux à trois minutes de disséquer une paire de ganglions. </li><li> Utilisez la pince pour couper et enlever le muscle d&#39;un côté d&#39;abord. </li><li> Une fois que le muscle est enlevé, l&#39;artère carotide deviendra visible. Souvent, il est léger rosé et contient du sang. Il peut apparaître plus clair que d&#39;autres vaisseaux sanguins que vous pouvez voir dans les environs et pour cette raison l&#39;artère carotide ne peut pas être perceptible immédiatement. Cette artère longe la trachée et se divise en ce qui ressemble à un &quot;Y&quot; en forme de ruban. Cette bifurcation est un jalon important et une fois il se trouve, il sera relativement facile de trouver la CTB. </li><li> Sever de l&#39;artère à l&#39;extrémité la plus caudale et soulevez-le légèrement avec la pince (l&#39;autre extrémité est encore attaché). Une fois que vous faites cela, vous verrez une forme d&#39;amande, brillant de masse à la recherche de tissu qui est lâchement à l&#39;artère juste au point de bifurcation et qui a thread-comme pré-et / ou post-ganglionnaires connectifs. C&#39;est le ganglion cervical supérieur. Utilisez une deuxième paire de pince dans l&#39;autre main au détachez qu&#39;elle et sa place dans une boîte de culture de petite taille (35 mm) contenant le froid, sans sérum du milieu RPMI 1640. </li><li> Ensuite, extraire le ganglion de l&#39;autre côté de la trachée suivant le même protocole. </li></ol> 3. Nettoyage les ganglions: <ol><li> Une fois que tous les ganglions ont été disséquées, ils devraient être libérés, autant que possible des tissus étrangers. </li><li> Sous le microscope de dissection, vous pouvez voir que les ganglions ont attaché des morceaux de l&#39;artère carotide, le tissu adipeux ou autres. Travailler avec une pince dans chaque main pour taquiner l&#39;écart de ces matières indésirables. Placez nettoyé ganglions dans une stérile conique 15ml, tube en polypropylène, contenant du milieu RPMI 1640. </li><li> Les connecteurs pré-et / ou post-ganglionnaires doivent également être coupé. Chaque ganglion a environ 10.000 neurones. Une fois que les neurones sont plaqués, ils vont repousser leurs neurites. </li></ol> 4. Établir la culture des neurones sympathiques: <ol><li> Centrifuger les ganglions au 200xg pendant 2 minutes et jeter le surnageant. </li><li> Resuspendre les ganglions de 0,5 à 1 ml de solution de trypsine à 0,25%. Placer dans un bain-marie 37 ˚ C ou un incubateur pendant 30 minutes. Cela aidera à dissocier les cellules dans les ganglions. </li><li> Après 30 minutes, ajouter 10 ml de milieu complet pour diluer et neutraliser la trypsine et centrifuger à 200xg pendant 2 minutes. </li><li> Rejeter le surnageant et remettre en suspension dans 2 ml de milieu final. </li><li> De plus dissocier les cellules par trituration des ganglions de haut en bas avec le feu-pipette en verre poli pâturage. Triturer les ganglions 20 fois et placer le tube sur la glace pendant quelques minutes. </li><li> Pendant ce temps, le feu-polonais le plus pipette pour faire l&#39;ouverture étroite (environ 2 / 3-1 / 2 mm de diamètre). Attendez quelques minutes jusqu&#39;à ce qu&#39;elle se refroidit et se triturer les ganglions 20 fois plus. Comme vous continuez, le milieu devient progressivement plus nuageux en indiquant que les cellules sont dissociées. Répétez cette étape une ou deux fois plus en fonction de la façon dont les cellules se dissocient. Après quelque temps, vous remarquerez que tous les ganglions sont dissociés en neurones et aucun ganglions intacts sont visibles dans le tube. Il peut y avoir du matériel tissulaire qui se dissocient pas. À ce stade, laissez reposer le tube dans la glace pendant quelques minutes afin que tous les débris non dissocié peut se déposent au fond. Maintenant recueillir soigneusement la quasi-totalité du milieu du haut et les placer dans un autre tube. Afin de s&#39;assurer que vous ne percevez pas les débris non dissocié, laissez environ 50 ul au fond du tube. Ajouter plus de la moyenne finale au tube contenant des cellules dissociées: ce volume dépend du type de culture est une plaque à l&#39;aide à la culture des neurones. Par exemple: si un plat de 24 puits doit être employé, puis la plaque 0,5 ganglions par puits (~ 10 000 neurones par puits) dans 0,5 ml de milieu final par puits. Une portée de rats typique se compose de 12 petits, ce qui donnerait 24 ganglions. Cela est suffisant pour un 24 graines bien-plaque et le volume total du milieu final est de 12 ml. Note: les ganglions contiennent également une petite population de cellules gliales et les autres types de cellules qui seront présents dans la culture. Utiliser Uridine/5-FDU dans le milieu final permettra d&#39;éviter leur prolifération et de promouvoir leur mort. </li><li> Flux cellules toutes les 48 heures en remplaçant 2 / 3 de la moyenne avec RPMI frais 1% de sérum de cheval + NGF et uridine/5-FDU. Remarque: Dans un premier temps, la culture contient des neurones sympathiques qui ont l&#39;air pas rond avec neurites. Neurites courts deviennent visibles 24 heures après la dissection et deviennent progressivement plus dense et plus ramifiée au cours du temps. </li></ol>

<ol>
	<li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , (1998).</li><li>Biswas, S. C., Shi, Y., Sproul, A., Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-apoptotic+Bim+Induction+in+Response+to+Nerve+Growth+Factor+Deprivation+Requires+Simultaneous+Activation+of+Three+Different+Death+Signaling+Pathways.">Pro-apoptotic Bim Induction in Response to Nerve Growth Factor Deprivation Requires Simultaneous Activation of Three Different Death Signaling Pathways.</a> The Journal of Biological Chemistry. 282 (40), 29368-29374 (2007).</li><li>Bocchini, V., Angeletti, P. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nerve+Growth+Factor:+purification+as+a+30,000-molecular-weight+protein.">The Nerve Growth Factor: purification as a 30,000-molecular-weight protein.</a> Proc. Natl. Acad. Sci. USA. (64), 787-794 (1969).</li><li>Dechant, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chat+in+the+Trophic+Web:+NGF+Activates+Ret+by+Inter-RTK+Signaling.">Chat in the Trophic Web: NGF Activates Ret by Inter-RTK Signaling.</a> Neuron. 33 (2), 156-158 (2002).</li><li>Deckwerth, T., Elliott, J. L., Knudson, C. M., Johnson, E. M., Snider, W. D., Korsmeyer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bax+is+required+for+neuronal+death+after+trophic+factor+deprivation+and+during+development.">Bax is required for neuronal death after trophic factor deprivation and during development.</a> Neuron. (17), 401-411 (1996).</li><li>Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+Vitro+Studies+on+the+Nerve+Growth+Factor+(NGF)+Requirement+of+Neurons+-+I.+Sympathetic+Neurons.">Quantitative in Vitro Studies on the Nerve Growth Factor (NGF) Requirement of Neurons - I. Sympathetic Neurons.</a> Developmental Biology. (58), 96-105 (1977).</li><li>Park, D. S., Morris, E. J., Padmanabhan, J., Shelanski, M. L., Geller, H. M., Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin-dependent+kinases+participate+in+death+of+neurons+evoked+by+DNA-damaging+agents.">Cyclin-dependent kinases participate in death of neurons evoked by DNA-damaging agents.</a> The Journal of Cell Biology. 143, 457-467 (1998).</li><li>Pierchala, B. A., Ahrens, R. C., Paden, A. J., Johnson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+Growth+Factor+Promotes+the+Survival+of+Sympathetic+Neurons+through+the+Cooperative+Function+of+the+Protein+Kinase+C+and+Phosphatidylinositol+3-Kinase+Pathways.">Nerve Growth Factor Promotes the Survival of Sympathetic Neurons through the Cooperative Function of the Protein Kinase C and Phosphatidylinositol 3-Kinase Pathways.</a> The Journal of Cell Biology. 279, 27986-27993 (2004).</li></ol>

The authors have nothing to disclose.

Nous utilisons des rats Sprague-Dawley pour nos cultures. Prenez le temps et les soins lors du nettoyage des ganglions. Enlever tous les débris, les vaisseaux sanguins et de graisse. Cette étape est importante pour assurer une culture qui est plus ou moins homogènes et de types de cellules étrangères. L&#39;ajout de uridine/5-FDU éliminera largement toutes les cellules restantes non-neuronales (mitose) contaminent la culture ou au moins supprimer leur prolifération. En outre, durant l&#39;étape de dissociation, être...

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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>Forceps, Stainless steel #5</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS4976</td>
<td>&nbsp;</td>
<tr>
<td>Scissors, DEAVER straight sharp-blunt- 43mm blades - 5.5</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS6760</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640 w/ L-Glutamin</td>
<td>Reagent</td>
<td>Cellgro, Mediatech, Inc.</td>
<td>10-040-CV</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>Reagent</td>
<td>SAFC Biosciences</td>
<td>12103c</td>
<td>&nbsp;</td>
<tr>
<td>Donor Horse Serum</td>
<td>Reagent</td>
<td>JRH Biosciences</td>
<td>12449</td>
<td>Heat-inactivate by incubating in 56&deg;C waterbath for 30 minutes.</td>
</tr>
<tr>
<td>Penicillin/streptomycin</td>
<td>Reagent</td>
<td>Gibco, Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin without EDTA</td>
<td>Reagent</td>
<td>Difco</td>
<td>MT 25-050-CI</td>
<td>&nbsp;</td>
<tr>
<td>Uridine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>U3003</td>
<td>&nbsp;</td>
<tr>
<td>5-Fluoro-5' deoxyuridine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>F-8791</td>
<td>&nbsp;</td>
<tr>
<td>NGF</td>
<td>Reagent</td>
<td>Harlan Bioproduct</td>
<td>BT-5025</td>
<td>&nbsp;</td>
<tr>
<td>Dissection Microscope and light source</td>
<td>Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>15 ml polypropylene tubes</td>
<td>Tool</td>
<td>BD Falcon</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>Cell culture dishes</td>
<td>Tool</td>
<td>Corning, Nunclone</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

protocol for culturing sympathetic neurons from rat superior cervical ganglia (scg)

Les ganglions cervicaux supérieurs (GCS) chez les rats sont petites, brillant, en forme d&#39;amande structures qui contiennent des neurones sympathiques. Ces neurones fournissent innervations sympathiques pour la tête et du cou et ils constituent une population bien caractérisés et relativement homogène (4). Neurones sympathiques sont tributaires du facteur de croissance nerveuse (NGF) pour la croissance de survie, la différenciation et axonale et la généralisation de la disponibilité du NGF facilite leur culture et leur manipulation expérimentale (2, 3, 6). Pour ces raisons, des cultures de neurones sympathiques ont été utilisés dans un large éventail d&#39;études, y compris le développement neuronal et la différenciation, les mécanismes de mort cellulaire programmée et pathologiques, et la transduction du signal (1, 2, 5 et 6). Disséquer la CTB à partir de rats nouveau-nés et les neurones sympathiques en culture n&#39;est pas très compliqué et peut être maîtrisé assez rapidement. Dans cet article, nous allons décrire en détail comment disséquer la CTB de ratons nouveau-nés et à les utiliser pour établir des cultures de neurones sympathiques. L&#39;article décrit également les étapes préparatoires et les différents réactifs et du matériel qui sont nécessaires pour atteindre cet objectif.

Watch this Scientific Journal Video about Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG at JoVE.com

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

C&#39;est un protocole décrivant la façon d&#39;isoler et de culture primaire de neurones sympathiques des ganglions cervicaux supérieurs (GCS) de ratons nouveau-né.

Protocole pour les neurones sympathiques de rat Culture ganglions cervicaux supérieur (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

protocol-for-culturing-sympathetic-neurons-from-rat-superior-cervical

Research

JoVE Journal

Biology

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

22.6K vues.  Columbia University. C'est un protocole décrivant la façon d'isoler et de culture primaire de neurones sympathiques des ganglions cervicaux supérieurs (GCS) de ratons nouveau-né.

Vidéo: Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

Préparation E18 neurones de rat corticale compartimentation dans un dispositif microfluidique

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

Recherche

Biologie

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

Isolement des neutrophiles Protocole

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

Préparation et entretien des neurones ganglions de la racine dorsale dans les cultures à compartiments

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

Une introduction au laboratoire Worm: La culture de Worms à la mutagenèse

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

Un protocole simple pour extraire l&#39;hémocytes de chenilles sauvages

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

Culture primaire Rat collecteur médullaire interne les cellules des canaux

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

L&#39;échantillonnage de sang de la veine caudale latérale de la Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Isolement de CD146<sup&gt; +</sup&gt; Cellules Resident pulmonaire mésenchymateuses stromales de Rat Poumons

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Un protocole simple à haut rendement pour l'isolement des îlettes pancréatiques des souris

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergstr&#246;m technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

This technical report describes a variation of the modified Bergstr&#246;m technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

Collection de biopsies musculaires squelettiques du compartiment supérieur du musculus humain tibialis antérieur pour l’évaluation mécanique

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...