Name: preparation of aplysia sensory-motor neuronal cell cultures
Uploaded: 2009-06-08T00:00:00
Description: Watch this Scientific Journal Video about Preparation of Aplysia Sensory-motor Neuronal Cell Cultures at JoVE.com

Die Arbeit im Labor mit der Kultivierung von Aplysia ist nach Liste NIH R01 und NIH R21MH077921 finanziert.

Vorbereitung (siehe Lösungen Abschnitt am Ende des Protokolls für die Zusammensetzung von Lösungen) <ol><li> Bereiten Kulturschalen. Beschichtung von Glas und von Mattek Glasboden-Kulturschale mit Poly-L-Lysin (made in Natriumborat). Add genug, um vollständig zu bedecken das Glas gut und lassen für&gt; 1 Std. (über Nacht bleiben). Gründlich von Poly-L-Lysin durch Spülen in künstlichem Meerwasser (ASW) 4-5 mal. Nach dem Entfernen der letzten Spülgang, fügen Sie 2 ml 50% L15 (ergänzt mit Salzen und mit L-Glu...

<ol>
	<li>Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+biology+of+memory+storage:+a+dialogue+between+genes+and+synapses.">The molecular biology of memory storage: a dialogue between genes and synapses.</a> Science. 294 (5544), 1030-1038 (2001).</li><li>Rayport, S. G., Schacher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+plasticity+in+vitro:+cell+culture+of+identified+Aplysia+neurons+mediating+short-term+habituation+and+sensitization.">Synaptic plasticity in vitro: cell culture of identified Aplysia neurons mediating short-term habituation and sensitization.</a> J Neurosci. 6 (3), 759-763 (1986).</li><li>Goldberg, D. J., Schacher, S., 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. , 213-236 (1998).</li><li>Glanzman, D. L., Kandel, E. R., Schacher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identified+target+motor+neuron+regulates+neurite+outgrowth+and+synapse+formation+of+aplysia+sensory+neurons+in+vitro.">Identified target motor neuron regulates neurite outgrowth and synapse formation of aplysia sensory neurons in vitro.</a> Neuron. 3 (4), 441-450 (1989).</li><li>Lyles, V., Zhao, Y., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapse+formation+and+mRNA+localization+in+cultured+Aplysia+neurons.">Synapse formation and mRNA localization in cultured Aplysia neurons.</a> Neuron. 49 (3), 349-356 (2006).</li><li>Martin, K. C., Casadio, A., Zhu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapse-specific,+long-term+facilitation+of+aplysia+sensory+to+motor+synapses:+a+function+for+local+protein+synthesis+in+memory+storage.">Synapse-specific, long-term facilitation of aplysia sensory to motor synapses: a function for local protein synthesis in memory storage.</a> Cell. 91 (7), 927-938 (1997).</li><li>Guan, Z., Giustetto, M., Lomvardas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+long-term-memory-related+synaptic+plasticity+involves+bidirectional+regulation+of+gene+expression+and+chromatin+structure.">Integration of long-term-memory-related synaptic plasticity involves bidirectional regulation of gene expression and chromatin structure.</a> Cell. 111 (4), 483-493 (2002).</li></ol>

Die erfolgreiche Herstellung von Aplysia sensomotorische Kulturen hat eine etwas langsame Lernkurve, da es die Entwicklung der Feinmotorik mit Mikrodissektion und Manipulation einzelner Nervenzellen über ein Stereo-Mikroskop betrachtet verbundenen beinhaltet. Nach unserer Erfahrung dauert es ca. 1-3 Wochen Praxis zu gesunden isolierten sensorischen Neuronen in Kultur und eine zusätzliche 1-3 Wochen erhalten zu lernen, wie sensorische Neuronen mit motorischen Neuronen Paar. Wir routinemäßig vorzubereiten Kul...

Lösungen benötigt für die Kultur <ol><li> 0,35 M MgCl 2, bei Raumtemperatur gelagert, verwendet für die Anästhesie. </li><li> Poly-L-Lysin-Lösung, Sigma: P-1524, MW&gt; 300.000, in 0,1 M Natrium-Borat pH8.2 bis 0,5-Promillegrenze Lösung gemacht. Vortex gut und filtriert durch ein 0,22 um Filter zu sterilisieren, lagern Sie es bei 4C °. Nicht Frost-Tau. </li><li> L-15 Medium-Pulver (Leibovitz) (Sigma: L4386) mit den Salzen, wie unten zu 1 Liter ergänzt <table border="1" cellspacing="0" cellpadding="0"><tr><td> L-15-Pulver </td><td> 13.8g </td></tr><tr><td> NaCl </td><td> 15,4 g </td></tr><tr><td> D-Glucose </td><td> 6,24 g </td></tr><tr><td> MgSO 4 7H 2 0 </td><td> 6.45g </td></tr><tr><td> KCl </td><td> 350 mg </td></tr><tr><td> NaHCO 3 </td><td> 170 mg </td></tr><tr><td> MgCl 2 6H 2 O </td><td> 5,49 g </td></tr><tr><td> CaCl 2 · 2H 2 O </td><td> 1.43g </td></tr><tr><td> HEPES </td><td> 3.53g </td></tr></table> Add ddH20 auf 1 Liter. Der pH-Wert sollte ca. 7,4-7,5 werden, fügen Sie 10 ml 100X Pen / Strep-Lösung und durch einen 0,22 m Filter Filter-sterilisiert. Lagerung bei 4C0 nicht länger als 1 Monat. </li><li> Proteaseverdau Lösung: 1% Protease IX (1unit/mg) ist in L15 gemacht (ergänzt mit Salzen wie oben) oder in ASW unmittelbar vor dem Gebrauch, durch einen 0,22 m Millipore-Filter-sterilisiert. 5mls sollte genug für den Ganglien von zwei Tieren (sicherstellen, dass die Ganglien vollständig in Protease-Lösung getaucht). Sigma hat berichtet, dass sie einzustellen Verkauf Protease IX. Ein Ersatz-Protease ist: Dispase II (Roche Applied Science Katalog # 04942078001). </li><li> Kulturmedium. Unmittelbar vor der Vorbereitung Kulturen, Tauwetter ein 10 ml Aliquot Hämolymphe und mischen Sie es mit 10 ml L15 (ergänzt mit Salzen wie oben), um 20mls Kulturmedium zu machen. Add 200 ul 200 mM L-Glutamin, gut mischen und Verwendung zur Herstellung von Kulturen. Dieses Medium sollte frisch zubereitet jedes Mal Kulturen gemacht werden. </li><li> Künstlichem Meerwasser kann von Instant Ocean (Aquarium Systems, Mentor, OH) oder wie folgt vorgenommen werden: 450 mM NaCl, 10 mM KCl, 30 mM MgCl 2 (6H 2 O), 20 mM MgSO 4, 10 mM CaCl 2 (2H 2 O), 10 mM HEPES, mit pH-Wert eingestellt auf 7,4. </li></ol>

preparation of aplysia sensory-motor neuronal cell cultures

Das Nervensystem der Meeresschnecke Aplysia californica ist relativ einfach, bestehend aus etwa 20.000 Neuronen. Die Neuronen sind groß (bis zu 1 mm im Durchmesser) und identifizierbar sind, mit unterschiedlichen Größen, Formen, Positionen und Pigmentierungen, und die Zellkörper nach außen in fünf gepaart Ganglien im ganzen Körper des Tieres verteilt ausgesetzt. Diese Eigenschaften haben die Forscher um die Schaltung zugrunde liegenden spezifischen Verhaltensweisen in der Tier 1 abzugrenzen erlaubt. Die monosynaptische Verbindung zwischen sensorischen und motorischen Neuronen ist ein zentraler Bestandteil der Kiemenrückziehreflex in das Tier, eine einfache Abwehrreflex in dem das Tier zieht ihren Kiemen in Reaktion auf taktile Stimulation des Siphons. Dieser Reflex erfährt Formen des nicht-assoziativen und assoziativen Lernens, einschließlich der Sensibilisierung, Gewöhnung und klassische Konditionierung. Von besonderem Nutzen für das Studium der synaptischen Plastizität kann der sensorisch-motorischen Synapse in der Kultur wieder hergestellt werden, wo gut charakterisierten Reize zu entlocken Formen der Plastizität, die direkt korreliert in das Verhalten des Tieres 2,3 haben. Speziell erzeugt Anwendung von Serotonin eine synaptische Verstärkung, dass, abhängig von der Anwendung Protokoll, dauert Minuten (kurzfristige Erleichterung), Stunden (mittelfristige Erleichterung) oder Tagen (langfristige Erleichterung). Im Gegensatz dazu erzeugt die Anwendung der Peptid-Sender FMRFamid eine synaptische Schwächung oder Depression, die, abhängig von der Anwendung Protokoll von Minuten bis Tage (long-term depression) dauern kann. Die Größe der Neuronen ermöglicht wiederholte scharfe Elektrode Aufnahme der synaptischen Erregung über Zeiträume von Tagen zusammen mit Mikroinjektion von Expressionsvektoren, siRNAs und andere Verbindungen auf spezifische Signalwege und Moleküle Ziel und damit die molekularen und zellbiologischen Schritte, die Änderungen unterliegen in der synaptischen Effizienz. Ein weiterer Vorteil der Aplysia Kultur-System kommt von der Tatsache, dass die Neuronen Synapsen-Spezifität zeigen, in der Kultur 4,5. So müssen sensorischen Neuronen nicht bilden Synapsen mit sich selbst (autapses) oder mit anderen sensorischen Neuronen, noch bilden sie Synapsen mit Nicht-Zielorganismen identifiziert Motoneuronen in der Kultur. Die Krampfadern, Stätten der synaptischen Kontakte zwischen sensorischen und motorischen Neuronen, sind groß genug (2-7 Mikrometer im Durchmesser), damit Synapsenbildung (sowie Veränderungen der synaptischen Morphologie) mit Ziel-Motoneuronen auf das Licht mikroskopischer Ebene untersucht werden. In diesem Video zeigen wir jeden Schritt der Herstellung sensorisch-motorischen Neurons Kulturen, einschließlich Anästhesie erwachsenen und jugendlichen Aplysia, sezieren ihre Ganglien, Proteaseverdau der Ganglien, die Entfernung des Bindegewebes durch Mikrodissektion, Identifizierung der beiden sensorischen und motorischen Neuronen und Entfernung jeder Zelltyp durch Mikrodissektion, dem Plattieren der Motoneuronen, neben der sensorischen Neuronen und Manipulation der sensorischen Neuriten Kontakt mit dem kultivierten Motoneuronen bilden.

Watch this Scientific Journal Video about Preparation of Aplysia Sensory-motor Neuronal Cell Cultures at JoVE.com

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primäre Kulturen von<em&gt; Aplysia</em&gt; Senso-motorischen Neuronen ein Modell Vorbereitung auf das Studium Synapsenbildung und synaptische Plastizität<em&gt; In-vitro-</em&gt;. Dieses Video zeigt die Identifikation und Mikrodissektion der sensorischen und motorischen Neuronen aus<em&gt; Aplysia</em&gt; Ganglien sowie die Methoden zur Festlegung und Aufrechterhaltung senso-motorischen Neuronen in Kultur.

Vorbereitung der Aplysia Sensory-Motor neuronalen Zellkulturen

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

preparation-of-aplysia-sensory-motor-neuronal-cell-cultures

Research

JoVE Journal

Biology

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES-Zellen abgeleiteten neuroepithelialen Zellkulturen

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

Forschung

Biologie

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

Primäre neuronale Kulturen aus den Gehirnen von Late Stage Drosophila Puppen

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

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.

Vorbereitung der neuronalen Kulturen aus Midgastrula Bühne Drosophila Embryonen

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

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.

Vorbereitung von dissoziierten Maus kortikale Neuron Kulturen

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

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Neuronalen Zellkulturen von Aplysia für High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

Maus Epidermal Neural Crest Stem Cell (EPI-NCSC) Kulturen

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

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.

Herstellung von 2-dGuo-Behandelte Thymus Organkulturen

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

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.

Erstellung und Pflege von Spinalganglien Neuronen in Compartmented Kulturen

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

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Die Herstellung von primären hämatopoetischen Zellkulturen aus Knochenmark der Maus für die Elektroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

Herstellung von Ratten-Gehirn-Aggregate Kulturen für Neuron und Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...