Abkürzungen: ACC - Adrenal chromaffinen Zellen, E. Soln - Enzymlösung, E. DMEM - Enriched Dulbeccos Modified Eagle Medium <ol><li> Vorbereitung <ol><li> Add Papain auf die gewünschte Menge an E. Soln (40 Einheiten Papain / 1ml E. Soln.). </li><li> Aktivieren Papain mit Carbogen für etwa 15 Minuten auf dem Eis. Oxygenat Lockes Puffer auf Eis als auch. </li></ol></li><li> Zergliederung <ol><li> Verwenden Sie 8-10 Wochen Maus. </li><li> Vor Verdauung entfernen Sauerstoff Lockes Puffer und auf Eis stellen. </li><li> Siehe Video zum Präparieren Verfahren. </li><li> Einmal ausgeschnitten, mit Sauerstoff statt Drüsen in Lockes Puffer. </li></ol></li><li> Vorbereitung von Gland <ol><li> Entfernen Sie Fett aus Drüse. </li><li> Streifen Rinde ab Drüse. </li></ol></li><li> Enzymatische Verdauung <ol><li> Sterilfilter Sauerstoff Papain. </li><li> Machen Sie vier Pools des oxygenierten Papain auf eine Petrischale. <ol><li> 3 Pools sollte etwa 100uls werden. </li><li> 1 des Pools sollte etwa 400uls werden. </li></ol></li><li> Wash medullae durch Pools <ol><li> 1. bis der 3 100 ul-Pools. </li><li> Dann durch die 400ul Pool </li><li> Pippette 300ul E. Soln und Medulla Stücke aus 400ul Pool in Mikrozentrifugenröhrchen. </li><li> Qrap Parafilm auf Mikrozentrifugenröhrchen und in 37 ° C Wasserbad für 20min. </li><li> Denken Sie daran, mit Sauerstoff zu versorgen restlichen E. Soln mit Papain fortsetzen. </li><li> Nach 20 Minuten Sterilfilter mehr E. Soln. </li><li> Ersetzen Sie alte E. Soln Baden medullae mit neu gefiltert E. Soln. </li></ol></li></ol></li><li> Verreibungen <ol><li> Absaugen und entsorgen E. Soln. </li><li> Wash medullae in 1ml E. DMEM. </li><li> Absaugen und entsorgen E. DMEM. </li><li> Add 300ul E. DMEM und verreiben 20X mit 1ml Spitze. </li><li> Cut 200 &amp;mgr; l Spitze mit einer sterilen Rasierklinge. </li><li> Man reibt 5-10X mit Ausschnitt 200 &amp;mgr; l Spitze. </li><li> Discard E. DMEM Baden medullae Stücke. Medium enthält Schutt. </li><li> Add 300uls frischen E. DMEM in Stücke medullae. </li><li> Man reibt mit 200 &amp;mgr; l Spitze 20x </li><li> Pippette E. DMEM Mikrozentrifugenröhrchen withough medullae Stücke. Medium enthält freie ACC. </li><li> Add 300uls frischen E. DMEM in Stücke medullae. </li><li> Man reibt medullae mit einem 23,5 gauge Kanüle 20x. </li><li> Kombinieren E. DMEM aus beiden Verreibungen. Medullae Stücke sollten nicht anwesend sein und sollten nun eine federähnliche Aussehen. </li></ol></li><li> Plating <ol><li> Microfuge E. DMEM aus den letzten beiden Verreibung Schritte für 2,5 Minuten bei 3,7 g s. </li><li> Pellet in 50uls-200 E. DMEM, abhängig von nachfolgenden Experimenten. </li><li> Platte 10-30uls auf einem Deckglas. </li><li> Warten Sie 15 Minuten für die Zellen zu haften. </li><li> Add 750uls von E. DMEM. </li></ol></li></ol>

The authors have nothing to disclose.

mouse adrenal chromaffin cell isolation

Nebennierenmarks chromaffinen Zellkultur-Systeme sind äußerst nützlich für die Untersuchung der Anregungs-Sekretion Kopplung in einem in vitro-Einstellung. Dieses Protokoll stellt das Verfahren verwendet werden, um den Nebennieren zu sezieren und dann isolieren medulläre Region durch Abstreifen der Nebennierenrinde. Die Verdauung der Medulla in einzelne chromaffinen Zellen wird dann demonstriert.

Watch this Scientific Journal Video about Mouse Adrenal Chromaffin Cell Isolation at JoVE.com

Mouse Adrenal Chromaffin Cell Isolation

Maus Adrenal Chromaffin Zellisolation

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This ...

Research

JoVE Journal

Biology

17.0K Aufrufe.  University of California, Irvine (UCI). Note: This video does n

Serie: Mouse Adrenal Chromaffin Cell Isolation

Mouse Dorsal Forebrain Explant Isolation

This video demonstrates the protocol for isolating and culturing explants of the mouse forebrain from embyonic day 12 mice. Procedures for removal of the uterus, embryos from uterus, and dissection of embryos are given. In addition the methodology for transferring these explants onto specialized membranes on which they are cultured is demonstrated. The development of the forebrain can be studied in vitro using this preparations as well as changes in gene expression.

Maus Dorsal Vorderhirnneuronen Explantation Isolation

Forschung

Biologie

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

Isolierung und Kultivierung von Post-Natal-Maus Cerebellar Granule Neuron Vorläuferzellen und Neuronen

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Isolation und Ableitung von embryonalen Maus-Keimzellen

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In-vivo-Imaging und therapeutische Behandlungen in einem orthotopen Mausmodell des Eierstockkrebs

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Isolierung von Maus Speicheldrüsen Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation &amp; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Isolation und Charakterisierung von Hoechst Low CD45 Negative Mouse Lung mesenchymalen Stammzellen

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generation der Mäuse aus induzierten pluripotenten Stammzellen

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Isolation und physiologische Analyse von Maus Kardiomyozyten

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Isolation und Kultur der erwachsenen Maus Kardiomyozyten für Cell Signaling und<em&gt; In-vitro-</em&gt; Herzhypertrophie

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal Glucocorticoid-Analyse: Nicht-invasive Monitoring-Adrenal in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...