Vorremmo ringraziare Katharina Cosker e Stephanie Courchesne per utili discussioni.

Preparazione dei reagenti <ol><li> Rivestimento collagene: collagene cappotto p35 piastre di coltura dei tessuti e mettere in forno a 37 ° C per 2 giorni prima ingrassaggio i divisori. La concentrazione finale di collagene deve essere di 0,71 mg / ml diluito in 0,001 N HCl. Poi, aggiungere 1 ml di miscela per piastra. </li><li> Caricatori Grasso: Per riempire il caricatore grasso, una siringa da 60 ml deve prima essere riempita con grasso per vuoto Corning. Usare la siringa per riempire il caricatore grasso, avvolgetela in un foglio e poi in autoclave per 45 minuti. </li><li> Divisori teflon: i divisori possono essere riutilizzati dopo ogni esperimento, ma deve prima essere puliti. Rimuovere il divisore dalla piastra, togliete tutti il ​​grasso residuo e il luogo in acido solforico per 2 giorni. Dopo aver rimosso dal lavaggio, sciacquare con acqua 3X, far bollire per 20 minuti, lasciare asciugare, mettere in un piatto di vetro P100 Petri e autoclave per 20 minuti. </li><li> N2-metilcellulosa: Pesare 1,5 g di metilcellulosa e mettetelo in una bottiglia da 500 ml. Aggiungi un ancoretta e autoclave per 20 minuti a secco (da questo punto tutto il lavoro deve essere sterile). Successivamente, aggiungere 500 ml di siero multimediale gratuito (N 2), e mescolate in una stanza fredda fino a quando non si scioglie. Aliquota in 50 conicals ml e congelare a -20 ° C. Per lavorare stock, aliquota uno dei 50 conicals mL in provette 1 ml e congelare a -20 ° C. </li><li> DRG media: DMEM, 5% di calore siero di cavallo inattivato, streptomicina e 1% di penicillina. </li><li> 100ng/mL DRGN dei media: la concentrazione stock di entrambi fattore di crescita nervosa (NGF) e del cervello-derivato fattore neurotrofico (BDNF) è 1mg/mL. Diluire ciascuna delle neurotrofine 1:10.000 nei media DRG. Culture possono essere coltivate in NGF solo: questo altera il complemento di neuroni che sopravvivono nelle culture. Nota: Quando necessario, la concentrazione di (citosina arabinoside) AraC è 1um e utilizzato ad una concentrazione finale di 0.3uM. Questo inibire la crescita di cellule di Schwann e glia altri. </li><li> 10ng/ml DRGN media: Diluire il 100ng/mL DRGN mezzi di comunicazione (1:10) con i media DRG. <img src="/files/ftp_upload/old/951_2008_10_27_6/951_Figure-01.png" alt="Figura 1" width="392" height="294" /> Strumenti di Figura 1. Necessarie per set-up </li></ol> La creazione di camere di compartimenti (avviare questo processo 1-2 giorni prima della dissezione) <ol><li> Fai un graffio nel bel mezzo di un piatto rivestito di collagene p35 con un movimento verso l&#39;esterno. </li><li> 30ul posto di N2-metilcellulosa al centro del graffio. Set piatti da parte fino divisore è ingrassato. </li><li> Allegare una 23-gauge adattatore luer stub al caricatore grasso. Afferrare il divisore in teflon con un paio di hemostats angolo di 90 ° e adagiarlo orizzontalmente con il divisore rivolta verso l&#39;alto sotto un microscopio. Traccia il divisore con grasso facendo in modo che ogni volta che l&#39;adattatore è posto a un nuovo punto di partenza è inserito l&#39;adattatore nella grasso dal passo precedente in modo che ci sia una linea continua di grasso (vedi schema). Una volta che il grasso viene applicato il divisore intero, girare uno dei piatti preparati p35 a testa in giù e posizionarlo in modo che il N2-metilcellulosa si trova sopra il vano centrale. Premere sul fondo del piatto con un paio di pinzette. Assicurarsi di premere sulla parte interna del divisore in quattro angoli (in alto a sinistra, in basso a destra, in alto a destra, in basso a sinistra, indicato nello schema da &quot;X&quot;). <img src="/files/ftp_upload/old/951_2008_10_9_2/951_Figure-02.png" alt="Figura 2" width="171" height="183" /> Figura 2: Procedura per ingrassare il divisore Nota: è importante per premere con forza sufficiente in modo che il grasso fa un sigillo completo con il piatto, ma se troppa pressione si aggiunge, gli assoni non attraverserà negli scompartimenti laterali. Sollevare il hemostats, capovolgerlo e sbloccare il divisore. Infine, posto il piatto con il divisore saldamente sotto il microscopio di messa a fuoco sul fondo del vano centrale. Con il caricatore grasso, fare una piccola barriera (0,25 centimetri) in modo che una volta che le cellule si trovano nel vano centrale non possono fuoriuscire. </li><li> Dopo aver creato diverse culture, media luogo DRG in ciascuno dei compartimenti laterali e posto in una incubatrice in cui le cellule saranno mantenute. Lasciare le culture di sedersi per diverse ore e poi verificare la presenza di perdite. Se il supporto è trapelato nel vano centrale, quindi la cultura è inutilizzabile. Nota: La prima volta che questa tecnica di apprendimento, è importante impostare le culture più di quelli necessari per un esperimento, come molti si perde. <img src="/files/ftp_upload/old/951_2008_10_9_3/951_Figure-03.png" alt="Figura 3" width="254" height="191" /> Figura 3: la cultura &quot;Good vs leaky&quot; </li></ol> Mantenere i neuroni DRG nelle culture compartimenti <ol><li> Giorno 1: Sostituire DRG media negli scomparti laterali con 100ng/mL DRGN supporti + AraC. Eseguire la dissezione e aggiungere le celle a scomparto centrale (100.000 cellule). </li><li> Giorno 2: Aggiungi 10ng/ml media + AraC verso l&#39;esterno del divisore Teflon fino a quando il flusso dei media oltre la barriera grasso e liquidi gli scambi con il vano centrale. </li><li> Giorno 3: Sostituire i media nei compartimenti laterali per 100ng/mL DRGN omettendo il Arac e il surround con 10ng/ml DRGN omettendo il AraC. </li><li> Giorno 6: sostituire i media nei compartimenti laterali per 1ng/mL + Arac e il surround con DRG supporti + AraC. </li><li> Giorno 9: Usa per la sperimentazione. <img src="/files/ftp_upload/old/951_2008_10_9_5/951_Figure-04.png" alt="Figura 4" width="267" height="148" /> Figura 4: immagini IHC di corpi cellulari e gli assoni distali Nota: Quando si cambiano i media, è importante per aspirare il liquido dalla parte superiore di ogni singolo vano laterale. Inoltre, non cambia il contenuto del vano centrale stessa, solo dal surround, e lasciate fluire oltre la barriera del grasso verso il centro. </li></ol>

<ol>
	<li>Campenot, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+Control+of+the+Local+Environment+of+Somas+and+Neurites.">Independent Control of the Local Environment of Somas and Neurites.</a> Methods in Enzymology. 58, 302-307 (1979).</li><li>Watson, F. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+use+the+Erk5+pathway+to+mediate+a+retrograde+survival+response.">Neurotrophins use the Erk5 pathway to mediate a retrograde survival response.</a> Nature Neuroscience. 4, 981-988 (2001).</li><li>Heerssen, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynein+motors+transport+activated+Trks+to+promote+survival+of+target-dependent+neurons.">Dynein motors transport activated Trks to promote survival of target-dependent neurons.</a> Nature Neuroscience. 7, 596-603 (2004).</li><li>Taylor, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nature Methods. 2, 599-605 (2006).</li><li>Park, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research.">Microfluidic culture platform for neuroscience research.</a> Nat Protoc. 4, 2128-2136 (2006).</li><li>Ure, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+transport+and+steady-state+distribution+of+125I-nerve+growth+factor+in+rat+sympathetic+neurons+in+compartmented+cultures.">Retrograde transport and steady-state distribution of 125I-nerve growth factor in rat sympathetic neurons in compartmented cultures.</a> J Neuroscience. 4, 1282-1290 (1997).</li></ol>

In questo video, abbiamo dimostrato come preparare e mantenere camere compartimenti per l&#39;uso nei neuroni DRG coltura. Fatto correttamente, questo sistema consente la separazione del corpo cellulare dal assone al fine di studiare meccanismi attraverso i quali neurotrofine segnale attraverso lunghi assoni. Poiché non vi è l&#39;isolamento fluidico tra i compartimenti, permette per la stimolazione selettiva o il trattamento di un compartimento senza altri compartimenti essere colpiti. Culture camera compartimentate in grado di supportar...

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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>collagen</td>
<td>Reagent</td>
<td>BD Biosciences</td>
<td>354249</td>
<td>&nbsp;</td>
<tr>
<td>N2-methylcellulose 400CPS</td>
<td>Reagent</td>
<td>Sel-Win Chemicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Teflon divider</td>
<td>Other</td>
<td>Tyler Research</td>
<td>CAMP10</td>
<td>many other types of dividers are available</td>
</tr>
<tr>
<td>Pin rake</td>
<td>Tool</td>
<td>Tyler Research</td>
<td>Camp-PR</td>
<td>&nbsp;</td>
<tr>
<td>Grease loader</td>
<td>Tool</td>
<td>Tyler Research</td>
<td>Camp-GLSS</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>Reagent</td>
<td>Fisher</td>
<td>MT10017CV</td>
<td>&nbsp;</td>
<tr>
<td>NGF</td>
<td>Reagent</td>
<td>Peprotech</td>
<td>450-01</td>
<td>&nbsp;</td>
<tr>
<td>BDNF</td>
<td>Reagent</td>
<td>Peprotech</td>
<td>450-02</td>
<td>&nbsp;</td>
<tr>
<td>High vacuum grease</td>
<td>Reagent</td>
<td>Fisher</td>
<td>14-635-5D</td>
<td>&nbsp;</td>
<tr>
<td>AraC</td>
<td>Reagent</td>
<td>Sigma</td>
<td>C-1768</td>
<td>&nbsp;</td>
<tr>
<td>23 gauge luer stub adapter</td>
<td>Tool</td>
<td>Fisher</td>
<td>427565</td>
<td>&nbsp;</td>
<tr>
<td>90&deg; angle hemostats</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS-7035</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

preparation and maintenance of dorsal root ganglia neurons in compartmented cultures

I neuroni estendere i processi assoni che sono molto lontane dal corpo cellulare a innervare tessuti bersaglio, dove sono richiesti obiettivi derivati ​​fattori di crescita per la sopravvivenza neuronale e la funzione. Neurotrofine sono specificamente tenuti a mantenere la sopravvivenza e la differenziazione dei neuroni sensoriali che innervano ma il problema di come questi target-derivati ​​neurotrofine comunicare al corpo cellulare dei neuroni che innervano è stata un&#39;area di ricerca attiva da oltre 30 anni. Il modello più comunemente accettata di come i segnali neurotrofina raggiungere il corpo cellulare propone di segnalazione endosomi portare questo segnale retrogrado lungo l&#39;assone. Al fine di studiare il trasporto retrogrado, un sistema di coltura è stato originariamente ideato da Robert Campenot, in cui corpi cellulari sono isolati dal loro assoni. La tecnica di preparazione di queste camere compartimenti per la coltura dei neuroni sensoriali ricapitola la stimolazione selettiva di terminazioni neuronali che si verifica in vivo seguente comunicato del target di derivazione neurotrofine. Retrograda di segnalazione eventi che richiedono lunga distanza retrograda dipendente microtubuli hanno importanti implicazioni per il trattamento di patologie neurodegenerative.

Watch this Scientific Journal Video about Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures at JoVE.com

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Qui si descrive la tecnica di preparazione e mantenimento di camere compartimenti per la coltura dei neuroni sensoriali dei gangli delle radici dorsali.

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 ...

preparation-maintenance-dorsal-root-ganglia-neurons-compartmented

Research

JoVE Journal

Biology

22.3K Views.  Dana Farber Cancer Institute. Qui si descrive la tecnica di preparazione e mantenimento di camere compartimenti per la coltura dei neuroni sensoriali dei gangli delle radici dorsali.

Video: Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Preparazione delle Culture neuronale da Midgastrula fase embrioni di Drosophila

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Ricerca

Biologia

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

Preparazione del dissociato Culture Neuron mouse corticale

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

Preparazione di proiettili pistola Gene e Biolistic Transfection dei neuroni in cultura Slice

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

La preparazione di 2-dGuo-trattati Organ Culture Timo

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

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

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4).  Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6).  For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6).  Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly.  In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons.  The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

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 ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Preparazione del Aplysia Senso-motorio colture cellulari neuronali

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Le colture per pluripotenti umane alternative staminali produzione di celle, manutenzione e analisi genetica

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

Laterale Root inducibile di sistema in<em&gt; Arabidopsis</em&gt; E mais

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

Divergenza del microbiota radicale in diversi habitat in base a reti di correlazione ponderate

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Un test di chemiotassi migliorato per la rapida identificazione dei chemioattrattivi rizobatterici negli essudati radicali

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...