NZ ringrazia laboratorio membri Subhas Biswas, Andrew Sproul, e Ryan Willet per la formazione lei in dissezione e la raccolta neuroni simpatici. Supportato anche da finanziamenti del NIH-NINDS.

1. Preparazione per la dissezione: <ol><li> Selezionare il piatto appropriata cultura (10 cm o piastre 6-24-bene o bene, ecc) a seconda della natura dei vostri esperimenti, e il cappotto con coda di topo collagene 24 ore prima della dissezione. I metodi per la preparazione di ratto coda di collagene e di utilizzarlo per i piatti della cultura cappotto sono stati descritti altrove (1). </li><li> Preparare una soluzione di tripsina 0,25% in tampone fosfato (PBS) (nessun EDTA) e filtro sterilizzare. Preparare 1 ml di aliquote e conservare a -20 ° C. </li><li> Preparare una soluzione contenente 2,5 mM ciascuno di uridina e 5-FDU in distillata / deionizzata H 2 O. Filtro-sterilizzare e preparare aliquote di 50 microlitri. Conservare a -20 º C. </li><li> Preparare Completa Media: terreno di coltura RPMI 1640 con 10% di siero siero di cavallo (calore inattivare incubando in un 56 º C bagnomaria per 30 minuti) e il 5% di siero fetale bovino. </li><li> Preparare finale Medio: terreno di coltura RPMI 1640 con 1% di siero siero di cavallo + NGF (concentrazione 100ηg/ml finale) + penicillina / streptomicina (1 / 250) + 1 / 250 soluzione uridine/5-FDU (10 microM concentrazione finale) . Nota: questo mezzo dovrebbero essere costituito fresca prima di utilizzare ogni volta. Per un esperimento in un 24-ben formato piatto, si dovranno preparare 12 ml di questo mezzo. Si raccomanda di preparare un piccolo extra. NGF può essere acquistato da una varietà di fonti commerciali (vedi tabella alla fine di questo protocollo). </li><li> Attrezzatura: Uno avrà bisogno di un microscopio da dissezione e fonte di luce. Un doppio illuminatore in fibra ottica a collo d&#39;oca con punte di messa a fuoco è preferito. Inoltre, si dovranno pulire e sterilizzare (in autoclave o immersione in etanolo al 70% per almeno 20 minuti) i seguenti strumenti di dissezione: due coppie di micro-dissezione pinze in acciaio inox, e un paio di forbici da dissezione (anche in acciaio inox e sia da Roboz). Due aghi da siringa sterile (26 gague x 1 ½ pollici o simili) sarà anche necessario. E &#39;necessario configurare un&#39;area dissezione, che è un 3 pezzi &quot;x3&quot; di polistirolo avvolto in un foglio di alluminio e ricoperta da una Kimwipe. Spruzzare la scheda con il 70% di etanolo per la decontaminazione. Infine, la preparazione del falò lucidato pascolo pipetta di vetro. Al fine di fuoco lucidare, prendere la pipetta di vetro e tenere la punta della fiamma per alcuni secondi, durante la rotazione. Al momento dell&#39;ispezione, la punta sarà più liscia e l&#39;apertura sarà più stretta. Ciò è necessario per il passaggio in cui le cellule saranno dissociate. Eseguire fuoco lucidatura in una cappa di coltura cellulare in modo che la pipetta rimane sterile. </li></ol> 2. Dissezione di Superiore gangli cervicali: <ol><li> Raccogliere cuccioli di ratto neonatale che sono di età P0 e P1. </li><li> Rapidamente strofinare il cucciolo per dissezione con il 70% di etanolo per ridurre la probabilità di contaminazione. </li><li> Rapidamente decapitare cucciolo con un paio di forbici affilate (questo passaggio non sarà mostrato nel video). Tenere la testa con pinza sterile contro garza sterile per drenare brevemente il sangue in eccesso. </li><li> Posizionare la testa sul pad dissezione con il lato ventrale rivolto verso l&#39;alto e la fine caudale orientata verso di voi. La trachea reciso deve essere facilmente visibile. Utilizzare gli aghi siringa sterile per proteggere la testa mozzata al pad dissezione nel modo seguente: spingere un pin anche se il tetto della bocca nel tappetino e spingere il secondo perno se reciso la trachea nel pad. Posizionare il cuscinetto sotto il microscopio da dissezione e iniziare la dissezione. </li><li> Utilizzando entrambe le pinze (una in ogni mano) la pelle chiara di distanza e grasso intorno alla trachea, facendo attenzione a non andare troppo in profondità. In questo modo si espongono un paio di muscoli correndo quasi parallele l&#39;una all&#39;altra su entrambi i lati della trachea. Nota: durante la dissezione, è necessario irrigare con acqua fredda PBS sterile o freddo, i livelli di free RPMI 1640 medium consegnato con una sterile, pipetta Pasteur per lavare via il sangue in eccesso e per mantenere l&#39;area pulita e umida. Ciò può essere fatto solo una volta o più volte a seconda di quanto velocemente si lavora. Per un dissettore esperienza, ci vogliono solo circa 2-3 minuti per sezionare fuori un paio di gangli. </li><li> Utilizzare le pinze per tagliare e rimuovere il muscolo da una parte per primo. </li><li> Una volta che il muscolo viene rimosso, l&#39;arteria carotide diventerà visibile. Spesso è la luce rosata e contiene sangue. Può apparire di colore più chiaro di altri vasi sanguigni che si può vedere nelle vicinanze e per questo motivo l&#39;arteria carotide può non essere evidente subito. Questa arteria costeggia la trachea e si biforca in quella che sembra una &quot;Y&quot; a forma di nastro. Questa biforcazione è un importante punto di riferimento e, una volta individuato, sarà relativamente facile trovare il SCG. </li><li> Recidere l&#39;arteria alla fine più caudale e sollevarla leggermente con la pinza (l&#39;altra estremità è ancora attaccato). Una volta fatto questo, vedrai una forma di mandorla, lucida massa alla ricerca di piccole di tessuto che è vagamente collegato al arteria proprio nel punto di biforcazione e che ha thread-come pre-e / o post-gangliari connettivi. Questo è il ganglio cervicale superiore. Usare un secondo paio di pinze con l&#39;altra mano per staccare delicatamente e mettere in un piatto cultura piccolo (35 mm) contenenti freddo, i livelli di free RPMI 1640 medium. </li><li> Successivamente, estrarre il ganglio sull&#39;altro lato della trachea seguendo lo stesso protocollo. </li></ol> 3. Pulizia dei gangli: <ol><li> Una volta che tutti i gangli sono state asportate, dovrebbero essere liberati il ​​più possibile di tessuti estranei. </li><li> Sotto il microscopio dissezione, si può vedere che i gangli hanno attaccato i pezzi di carotide, tessuto adiposo o altro. Lavora con pinze in ogni mano per prendere in giro via questi materiali indesiderati. Luogo pulito gangli in una sterile 15 ml conica, tubo di polipropilene, contenente RPMI 1640 medium. </li><li> I connettivi pre e / o post-gangliari deve essere tagliato via. Ogni ganglio ha circa 10.000 neuroni. Una volta che i neuroni sono placcati, saranno ricrescere i loro neuriti. </li></ol> 4. Stabilire cultura Simpatico Neuron: <ol><li> Centrifugare i gangli a 200xg per 2 minuti e scartare il surnatante. </li><li> Risospendere i gangli in 0,5 a 1 ml di soluzione tripsina 0,25%. Posto a 37 ˚ C a bagno-maria o incubatore per 30 minuti. Questo aiuterà dissociare le cellule nei gangli. </li><li> Dopo 30 minuti, aggiungere 10 ml di terreno completo per diluire fuori e neutralizzare la tripsina e centrifugare a 200xg per 2 minuti. </li><li> Gettare il surnatante e risospendere in 2 ml di media finale. </li><li> Ulteriori dissociare le cellule dei gangli triturazione su e giù con il fuoco lucidato pascolo pipetta di vetro. Triturare i gangli 20 volte e posizionare il tubo in ghiaccio per qualche minuto. </li><li> Nel frattempo, il fuoco, lucidare la pipetta di più per rendere l&#39;apertura più stretta (circa 2 / 3 a 1 / 2 mm di diametro). Attendere qualche minuto fino a quando si raffredda e triturare i gangli 20 volte di più. Come si continua, il medium diventa progressivamente più nuvolosa indicando che le cellule vengono dissociate. Ripetere questa operazione una o due volte più a seconda di quanto bene le cellule si dissociano. Dopo qualche tempo, noterete che tutti i gangli sono dissociate in neuroni dei gangli intatto e non sono visibili nel tubo. Ci possono essere alcuni materiali tessuto che non si dissociano. A questo punto, lasciare che il tubo di sedersi in ghiaccio per qualche minuto in modo che i detriti non dissociato possono depositarsi sul fondo. Ora attentamente raccogliere quasi tutti i media dalla parte superiore e luogo in un altro tubo. Per assicurarsi di non raccogliere i detriti non dissociato, lasciare circa 50 microlitri sul fondo della provetta. Aggiungere più del mezzo finale per il tubo contenente cellule dissociate: questo volume dipende dal tipo di targa una cultura sta usando per la cultura i neuroni. Ad esempio: se un 24-ben piatto è da utilizzare, poi piatto 0,5 gangli per bene (~ 10.000 neuroni per pozzetto) in 0,5 ml di terreno finale per pozzetto. Un disordine ratto tipico consiste di 12 cuccioli, che darebbe 24 gangli. Questo è sufficiente per seminare uno a 24 pozzetti e il volume totale dei media finale è di 12 ml. Nota: i gangli contengono anche una piccola popolazione di cellule gliali e altri tipi di cellule che saranno presenti nella cultura. Utilizzando Uridine/5-FDU nel medio finale impedisce la loro proliferazione e promuovere la loro morte. </li><li> Nutrire le cellule ogni 48 ore, sostituendo 2 / 3 del mezzo con fresco RPMI +1% siero di cavallo + NGF e uridine/5-FDU. Nota: In un primo momento, la cultura contiene neuroni simpatici che guardano intorno senza neuriti. Neuriti breve diventano visibili 24 ore dopo la dissezione e progressivamente diventano più densi e più ramificate nel tempo. </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.

Noi usiamo ratti Sprague Dawley per le nostre culture. Prendetevi il tempo e attenzione durante la pulizia dei gangli. Rimuovere tutti, detriti vasi sanguigni e grasso. Questo passo è importante per garantire una cultura che è più o meno omogeneo e privo di tipi di cellule estranee. L&#39;aggiunta di uridine/5-FDU in gran parte eliminare eventuali cellule rimanenti non neuronali (mitosi) che contaminano la cultura, o almeno sopprimere la proliferazione. Inoltre, durante la fase di dissociazione, di pazienza e il tempo di ...

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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)

I gangli cervicale superiore (SCG) nei ratti sono piccole, lucide, a mandorla strutture che contengono neuroni simpatici. Questi neuroni fornire innervazioni simpatico per la testa e del collo e costituiscono una popolazione ben caratterizzato e relativamente omogenea (4). Neuroni simpatici dipendono dal fattore di crescita nervosa (NGF) per la crescita sopravvivenza, differenziazione e assonale e la diffusa disponibilità di NGF facilita la loro cultura e la manipolazione sperimentale (2, 3, 6). Per queste ragioni, colture di neuroni simpatici sono stati utilizzati in una vasta gamma di studi tra cui lo sviluppo neuronale e la differenziazione, i meccanismi della morte cellulare programmata e patologiche, e trasduzione del segnale (1, 2, 5 e 6). Sezionare la SCG di ratto appena nati e dei neuroni simpatici coltura non è molto complicata e può essere imparato abbastanza rapidamente. In questo articolo, descriveremo in dettaglio come sezionare la SCG da cuccioli di ratto appena nati e di utilizzarle per stabilire le culture di neuroni simpatici. L&#39;articolo si descrivono anche le fasi preparatorie e reagenti vari e le attrezzature che sono necessarie per raggiungere questo obiettivo.

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

Questo è un protocollo che descrive come isolare e colture primarie di neuroni simpatici gangli cervicale superiore (SCG) di cuccioli di ratto appena nati.

Protocollo per la coltura neuroni simpatico da Rat gangli cervicale superiore (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 Views.  Columbia University. Questo è un protocollo che descrive come isolare e colture primarie di neuroni simpatici gangli cervicale superiore (SCG) di cuccioli di ratto appena nati.

Video: 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.

Preparazione E18 neuroni corticali di ratto per compartimentazione in un dispositivo a microfluidi

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

Ricerca

Biologia

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.

Neutrofili isolamento Protocollo

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.

Preparazione e manutenzione dei neuroni dei gangli dorsali in Culture Compartmented

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.

Introduzione al Laboratorio Worm: da Worms coltura a mutagenesi

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 protocollo semplice per Estrazione emociti da bruchi selvaggi

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.

Coltura primaria Rat midollare interna Raccolta cellule del dotto

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.

Prelievo di sangue dalla vena della coda laterale del Topo

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.

Isolamento di CD146<sup&gt; +</sup&gt; cellule residenti del polmone mesenchimali stromali da Rat Polmoni

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 semplice protocollo ad alta efficienza per l'isolamento delle isolozioni pancreatiche dai topi

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.

Collezione di biopsie muscolari scheletriche dal compartimento superiore del musolo umano Tibialis Anterior per la valutazione meccanica

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