Diese Arbeit wurde von der Deutschen Forschungsgemeinschaft (; KL 1415/3-2 und KL 1415/4-2 DFG) unterstützt.

1. Vorbereitung <ol><li> Vorbereitung der Kulturschalen <ol><li> Thaw Kollagen Typ IV O / N bei 4 ° C und löst ihn in 0,1% sterile Essigsäure. Das Volumen zu verwenden, hängt von der Art und Anzahl der Gerichte, in denen die Zellen ausgesät zu werden. Verwenden 2 pg / cm 2. </li><li> Inkubieren Gerichte mindestens 1 h bei RT und waschen zweimal mit destilliertem Wasser (A. tridest). </li><li> Lassen Sie die Gerichte, um richtig zu trocknen. </li></ol></li><li> Supplementation des Mediums <ol><li> Erhöhen der Blutzuckerspiegel bis zu 4,5 g / L, indem 1,75 g Glucose in 500 ml Medium. Um das Medium auf 600 mosmol einzustellen, mit 100 mM NaCl und 100 mM Harnstoff, um die Osmolarität von 200 mosmol und 100 mosmol erhöhen, beziehungsweise. </li><li> 1% nicht-essentielle Aminosäuren, 1% Ultroser, 500 pM DBcAMP, 20 U / ml Nystatin und 0,25 g / ml Gentamicin (1:200-Verdünnung der Stammlösung mit einer Konzentration von 50 ug / ml) auf 600 mosmol DMEM-Medium frisch am Tag der Vorbereitung. </li></ol></li&gt; <li> Enzymlösung <ol><li> 1 mg / ml Hyaluronidase und 2,2 mg / ml Collagenase-DPBS, enthaltend 0,25 g / ml Gentamicin und Nystatin. Bereiten 1 ml dieser Enzymlösung für die Verdauung von 2 Niere inneren medullae. Die Lösung muss durch Filtration sterilisiert werden und wird in 50 ml-Falcon-Röhrchen gelagert. </li></ol></li></ol> 2. Kultivierung von primären Ratten Inner Medulläre Sammelkanal (IMCD) Cells Alle tierischen Handhabung Verfahren sind in Übereinstimmung durchgeführt werden mit der lokalen und föderalen Gesetzgebung. <ol><li> Betäuben Ratten in CO 2 (in einer Box mit CO 2 gesättigt) oder Isofluran (U-400 Anästhesie-Einheit, Univentor Ltd, Malta; Luftstrom 350-400 ml / min mit 2-2,5% Isofluran). Enthaupten die Tiere für Blutungen, die genaue Erkennung der Grenze zwischen dem inneren und dem weißlich dunkleren äußeren Medulla erlaubt. Darüber hinaus minimiert die Blutung die Anzahl der isolierten Erythrozyten. Nieren und entfernenwaschen Sie sie in sterilen DPBS mit Gentamicin und Nystatin (siehe oben) auf Eis ergänzt. Alle weiteren Schritte sind unter sterilen Bedingungen saubere Bank durchgeführt werden. </li><li> Übertragen Nieren aus dem DPBS Lösung auf eine sterile Kompresse auf überschüssige Flüssigkeit zu entfernen. Entfernen Sie das Fett Niere Kapsel mit einer sterilen Pinzette und Schere. </li><li> Nachdem die Niere noch in der Kompresse befestigt ist, geschnitten etwas neben der Längsachse des äußeren (kortikalen) Seite. Achten Sie darauf, nicht durch völlig abgeschnitten, sondern um es in einem Stück zu verlassen, verbunden im Nierenbecken. </li><li> Mit gebogenen Schere sorgfältig sezieren die weißlich inneren medullae, die durch die rosé schimmernde äußeren Medulla umgeben sind. Sammeln Sie in DPBS, mit Gentamicin und Nystatin in einer 60 mm Kulturschale auf Eis. </li><li> Sobald alle medullae isoliert und gesammelt auf Eis, reduzieren Sie die Lautstärke von DPBS auf ein Niveau, wo sie gerade mit Puffer bedeckt sind. Verwenden Sie eine sterile Rasierklinge die medullae in Würfel schneiden von 5mm 3. </li><li> Schmelzen und die scharfe Ende einer sterilen Pasteurpipette indem es kurz in eine Flamme. Achten Sie darauf, die Pipette, bevor Sie fortfahren gekühlt wird. </li><li> Übertragen des Gewebes Suspension auf die 50 ml-Falcon-Röhrchen, die frisch hergestellten sterilen Enzymlösung mit Hyaluronidase und Kollagenase (siehe 1.3.1) unter Verwendung der gerundeten Pasteurpipette. Schließen Sie den Deckel fest und Inkubation bei 37 ° C unter ständigem Rühren (220 rpm) in einem regulären tabletop Wasserbad für 1,5 - 2 Stunden. </li><li> Zum Zerreiben der Suspension, Pipette das Gemisch nach oben und unten mehrmals mit dem runden Pasteurpipette (siehe oben), bis die Suspension homogen wird trüb. </li><li> Zentrifugieren Sie die Suspension für 5 min bei 300 xg und 16 ° C. Überstand verwerfen, das Pellet gründlich in DPBS, mit Gentamicin und Nystatin und Zentrifuge wieder. Wiederholen Sie diesen Schritt einmal waschen. </li><li> Die Zellen in voll ergänzt mittel-und Saatgut sie in culture Gerichte und / oder Mikro-Titer-Well-Platten. Die Herstellung von 2 medullae (1 Tier) wird genügend Zellen für eine 60 mm Kulturschale ergeben. </li><li> Nach 24 Stunden, waschen Sie die Zellen zweimal mit 600 mosmol DMEM und fügen Sie frisches Medium. Während der nächsten Woche sollten die Zellen 2-3 mal (Abbildung 1) gewaschen werden. 6-8 Tage nach der Aussaat können die IMCD Zellen für Experimente verwendet werden. Denken Sie daran, sie in Medium inkubieren ohne DBcAMP und Nystatin für 24 Stunden vor Beginn des Experiments. Ansonsten AQP2 wird ausschließlich in der Plasmamembran lokalisiert werden. </li></ol>

<ol>
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Die Autoren erklären, dass sie keine finanziellen Interessen haben.

Wir präsentieren ein detailliertes Protokoll für die Vorbereitung und Kultivierung von primären Ratten IMCD Zellen. Der Ansatz liefert bis zu 21 cm 2 von Zellen aus einer Ratte 20. Der Versuch erfordert Standard Zellkulturausrüstung und kann von einer einzigen Person innerhalb von etwa 6 Stunden durchgeführt werden. Daher ist dieser Ansatz eignet sich als Standard-Labormethode. Primäre Ratte IMCD Zellen in Kulturschalen unterschiedlicher Größe ausgesät werden, ...

A correction was made to <a href="http://www.jove.com/video/50366">Culturing Primary Rat Inner Medullary Collecting Duct Cells</a>. There was an error with an author's name. The author's last name had a typo and was corrected to: Enno Klussmann instead of: Enno Klussman

A correction to author list has been made for article Culturing Primary Rat Inner Medullary Collecting Duct Cells.

Erratum: Culturing Primary Rat Inner Medullary Collecting Duct Cells

In renalen Sammelkanal Hauptzellen steuert Arginin-Vasopressin (AVP) Reabsorption von Wasser durch die Stimulierung der Insertion des Wassers Aquaporin-2 (AQP2) in die Plasmamembran. AVP bindet an das G-Protein-gekoppelten Vasopressin-Typ-2-Rezeptor (V2R) stimuliert Adenylatcyclase und somit cAMP-Bildung. Initiation dieser Signalkaskade führt zur Aktivierung der Proteinkinase A (PKA). PKA phosphoryliert AQP2 an Serin 256 (S256), das ist der Schlüssel Auslöser für seine Umverteilung von intrazellulären Vesikeln in die Plasmamembran. Die Membran Einsetzen erleichtert Wasserreabsorption entlang eines osmotischen Gradienten und Feinabstimmung Körper Wasser-Homöostase. Dysregulation der AVP-vermittelten Wasserreabsorption aufgrund Aberrationen AVP-Sekretion oder AVP-aktivierten Signalisierung Ursachen oder ist mit schweren Erkrankungen. Verminderte Wasserreabsorption durch Mutationen des V2R oder AQP2 verursacht führt zu Diabetes insipidus, während eine elevATED Blutplasma AVP Ebene wird mit überschüssigem Wasser Reabsorption in Herz-Kreislauf-Erkrankungen wie chronischer Herzinsuffizienz und das Syndrom der inadäquaten ADH-Sekretion (SIADH) verbunden. Die Bedeutung der AVP-vermittelten Wasserreabsorption rührt nicht nur von seiner Verwicklung in Krankheit. Die AVP-induzierte Translokation von AQP2-tragenden Vesikeln und Fusion mit der Zellmembran eine streng cAMP-abhängige Exozytose-Verfahren, die gegenwärtig nicht gut verstanden. Weitere Beispiele für eine cAMP-abhängige Exozytose Reninsekretion in der Niere und der H +-Sekretion in den Magen. Daher Aufklärung der molekularen Mechanismen für die AQP2-Translokation in renalen Hauptzellen hilft nicht nur ähnliche molekulare Prozesse in anderen Zelltypen zu verstehen, sondern kann auch den Weg zu neuen Therapien für die Behandlung von Erkrankungen, die mit Störungen der AVP-vermittelten Wasserreabsorption verbunden . Elucidating Kontrollmechanismen AQP2 erfordert Sammelkanal Hauptzellen Modelle. Hierzu wird eine Anzahl von verschiedenen Säugetierniere Zelllinien sind. Jedoch haben diese Modelle mehrere Nachteile. In vielen Zellsysteme der Protein-Ebene von AQP2 niedrig ist (Tabelle 1; M1, HEK293, COS-7, MDCK, LLC-PK1) 1-10, andere ektopisch überexprimieren human (MCD4, WT10) 11,12 oder Ratte (CD8) 13 AQP2. In MCD4 und COS-7-Zellen, die V2-Rezeptor nicht exprimiert. Nach unserer Kenntnis gibt es keine permanente Zelllinie als Modell zur Verfügung, die von der Nierenfunktion inneren medullären Sammelkanal, dass ein Teil der Niere, wo AQP2 Expression häufigste abgeleitet ist, sondern von der kortikalen Sammelkanal 1,11,13-16 . Solche Zelle Modelle sind zum Beispiel das weit verbreitete verewigt mpkCCD (zB 17) oder die neu gegründete mTERT-CCD 14 Zelllinien. Beide Zelllinien endogen express AQP2 und V2R aber da sie aus der abgeleitetenkortikalen Sammelkanal wahrscheinlich eine unterschiedliche Proteom gegenüber inneren medullären Sammelkanal (IMCD-Zellen). Sie müssen zum Beispiel auszudrücken verschiedenen Na +-Transport-Systeme. Hier präsentieren wir ein Protokoll zur Kultur primären IMCD Zellen V2R und AQP2 endogen. Dieses Modell stellt daher am ehesten die physiologische Situation in der Nieren-Sammelkanal. Da die Gründung der Kultur erfordert nur Standard Laborgeräte anderen Laboratorien können leicht annehmen diesen Ansatz.

<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Company Catalog</td>
			<td>Number</td>
		</tr>
		<tr>
			<td>Collagen Type IV Mouse</td>
			<td>BD Biosciences</td>
			<td>356233</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>SIGMA</td>
			<td>H6254</td>
		</tr>
		<tr>
			<td>Collagenase type CLS-II</td>
			<td>Biochrom AG</td>
			<td>C2-22</td>
		</tr>
		<tr>
			<td>DMEM + GlutMAX</td>
			<td>Invitrogen GIBCO</td>
			<td>21885108</td>
		</tr>
		<tr>
			<td>Nystatin</td>
			<td>SIGMA</td>
			<td>N4014</td>
		</tr>
		<tr>
			<td>Ultroser G</td>
			<td>Cytogen</td>
			<td>15950-017</td>
		</tr>
		<tr>
			<td>Non essential amino acids (NEA)</td>
			<td>Biochrom AG</td>
			<td>K0293</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Invitrogen GIBCO</td>
			<td>15710</td>
		</tr>
		<tr>
			<td>DBcAMP</td>
			<td>BIOLOG</td>
			<td>D009</td>
		</tr>
	</tbody>
</table>

culturing primary rat inner medullary collecting duct cells

Arginin-Vasopressin (AVP) erleichtert Wasserreabsorption durch renale Sammelkanal Hauptzellen und damit eine Feinabstimmung Körper Wasser-Homöostase. AVP bindet Vasopressin V2-Rezeptoren (V2R) auf der Oberfläche der Zellen und induziert dadurch Synthese von cAMP. Dies stimuliert die zelluläre Signalgebung Prozesse, die zu Veränderungen in der Phosphorylierung des Wassers Aquaporin-2 (AQP2). Protein Kinase A phoshorylates AQP2 und löst dadurch die Translokation von intrazellulären Vesikeln AQP2 in die Plasmamembran zu erleichtern Reabsorption von Wasser aus primären Urin. Aberrationen der AVP-Freisetzung aus der Hypophyse oder AVP-aktivierten Signalisierung in Hauptzellen kann zentral oder Diabetes insipidus sind; eine erhöhte Blutplasma AVP Ebene ist mit Herz-Kreislauf-Erkrankungen wie chronischer Herzinsuffizienz und das Syndrom der inadäquaten ADH-Sekretion verbunden. Hier präsentieren wir ein Protokoll für cultivatiauf der primären Ratten inneren medullären Sammelkanal (IMCD-Zellen), die ausdrückliche V2R und AQP2 endogen. Die Zellen eignen sich zur Erläuterung molekularen Mechanismen, die der Steuerung der AQP2 und damit Entwicklung neuer Medikamente zur Behandlung von Erkrankungen mit der Fehlregulation der AVP-vermittelten Wasserreabsorption verbunden zu entdecken. IMCD Zellen aus Ratte renale inneren medullae erhalten und werden für Experimente sechs bis acht Tage nach der Aussaat verwendet. IMCD Zellen in regelmäßigen Zellkulturschalen, Flaschen und Mikrotiterplatten unterschiedlicher Formate können kultiviert werden, das Verfahren erfordert nur ein paar Stunden, und ist geeignet für Standard Zellkulturlaboren.

Die erfolgreiche Kultivierung von primären Ratten IMCD-Zellen werden in einem Monolayer 6-8 Tage nach der Aussaat (Abbildung 2) führen. Per 60 mm Kulturschale gibt es etwa 6 x 10 6 Zellen. Die Zellen eng an den Kulturschalen haften, da diese mit Kollagen Typ IV, eine Basalmembran Komponente 18 beschichtet wurden. Daher wird IMCD Zellen nicht einmal lösen während mehrerer gründliches Waschen Verfahren. Bis zu 80% der kultivierten Zellen exprimieren endogen V2R und AQP2. Dies si...

Watch this Scientific Journal Video about Culturing Primary Rat Inner Medullary Collecting Duct Cells at JoVE.com

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginin-Vasopressin (AVP) steuert Feinabstimmung der Körper Wasser Homöostase durch die Erleichterung Wasserreabsorption durch renale Hauptzellen. Hier präsentieren wir ein Protokoll für die Kultivierung von primären Ratten inneren medullären Sammelkanal Zellen geeignet für die Aufklärung der zugrunde liegenden molekularen Mechanismen AVP-vermittelten Wasserreabsorption.

Kultivierung primären Ratten Inner Medulläre Sammelkanal Cells

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

culturing-primary-rat-inner-medullary-collecting-duct-cells

Research

JoVE Journal

Biology

14.7K Aufrufe.  Max-Delbrück-Center for Molecular Medicine. Arginin-Vasopressin (AVP) steuert Feinabstimmung der Körper Wasser Homöostase durch die Erleichterung Wasserreabsorption durch renale Hauptzellen. Hier präsentieren wir ein Protokoll für die Kultivierung von primären Ratten inneren medullären Sammelkanal Zellen geeignet für die Aufklärung der zugrunde liegenden molekularen Mechanismen AVP-vermittelten Wasserreabsorption.

Serie: Culturing Primary Rat Inner Medullary Collecting Duct Cells

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.

Protokoll zur Kultivierung sympathischen Neuronen aus Ratten-Superior Halsganglien (SCG)

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

Forschung

Biologie

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Primäre Kultur der erwachsenen Ratte Herz Myozyten

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

Primären humanen bronchialen Epithelzellen aus Explantaten Grown

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

Isolierung von Basalzellen und Submuköse Gland Duct Zellen aus Maus Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Isolierung und Primärkultur von Ratten Leberzellen

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Isolierung von Immunzellen aus Primärtumoren

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

Die Kultivierung der menschlichen Nasen Epithelzellen an der Air flüssig-Grenzfläche

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

Ein Verfahren zur Kultivierung von embryonalen<em&gt; C. elegans</em&gt; Zellen

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-innere Zelle Masse Injektion von embryonalen Stammzellen führt zu höheren Chimerism Raten

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

Primärkultur von Ratte Nebennierenrindeninsuffizienz Zellen und Assays Steroidogenic Funktionen

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...