Ce travail a été soutenu par la Deutsche Forschungsgemeinschaft (DFG; KL 1415/3-2 1415/4-2 et KL).

1. Préparation <ol><li> Préparation des boîtes de culture <ol><li> Dégel collagène de type IV O / N à 4 ° C et le dissoudre dans l&#39;acide acétique stérile de 0,1%. Le volume à utiliser dépend du type et du nombre de plats dans lesquels les cellules doivent être ensemencées. Utilisation 2 ug / cm 2. </li><li> Incuber plats au moins pendant 1 heure à température ambiante et laver deux fois avec de l&#39;eau distillée (A. tridest). </li><li> Les laisser sécher correctement. </li></ol></li><li> La supplémentation en moyenne <ol><li> Augmentez le niveau de glucose à 4,5 g / L en ajoutant 1,75 g de glucose à moyen de 500 ml. Pour ajuster le milieu à 600 mosmol, ajouter du NaCl 100 mM et 100 mM d&#39;urée, pour augmenter l&#39;osmolarité de 200 mosmoles et 100 mOsmol, respectivement. </li><li> Ajouter 1% d&#39;acides aminés non essentiels, 1% Ultroser, 500 um DBcAMP, 20 U / ml nystatine et 0,25 pg / ml de gentamicine (1:200 dilution de la solution mère avec une concentration de 50 ng / ml) à 600 DMEM mosmol fraîchement le jour de la préparation. </li></ol></li&gt; <li> solution enzymatique <ol><li> Ajouter 1 mg / ml d&#39;hyaluronidase et 2,2 mg / ml de collagénase à DPBS contenant 0,25 mg / ml de gentamicine et la nystatine. Préparer 1 ml de cette solution enzymatique pour la digestion des 2 reins moelles intérieure. La solution doit être stérilisée par filtration et est stocké dans des tubes Falcon 50 ml. </li></ol></li></ol> 2. La culture du primaire Rat canal collecteur médullaire interne (IMCD) Cellules Toutes les procédures de manipulation des animaux doivent être réalisées dans le respect de la législation locale et fédérale. <ol><li> Anesthésier les rats de CO 2 (dans une boîte saturée de CO 2) ou à l&#39;isoflurane (U-400 appareil d&#39;anesthésie, Univentor Ltd, Malte, le débit d&#39;air de 350-400 ml / min avec 2-2,5% d&#39;isoflurane). Décapiter les animaux de saignement qui permet la détection précise de la frontière entre l&#39;intérieur blanchâtre et la médullaire externe plus sombre. En outre, le saignement réduit le nombre de globules rouges isolés. Retirer les reins etlavez-les dans du DPBS stérile additionné de gentamicine et la nystatine (voir ci-dessus) sur la glace. Toutes les autres étapes doivent être réalisées dans des conditions de banc propre stériles. </li><li> Transfert reins de la solution DPBS sur une compresse stérile pour enlever le liquide excessif. Retirez la capsule rénale gras avec pinces et de ciseaux stériles. </li><li> Ayant le rein encore fixé dans la compresse, coupé légèrement à côté de l&#39;axe longitudinal de la face externe (cortex). Veillez à ne pas couper à travers complètement, mais de le laisser dans une seule pièce, connecté dans le bassinet du rein. </li><li> Avec des ciseaux courbes disséquer soigneusement le moelles interne blanchâtre, qui sont entourés par la médullaire externe chatoyant rosé. Collectionnez-les dans du DPBS, contenant de la gentamicine et la nystatine dans une boîte de culture de 60 mm sur la glace. </li><li> Une fois tous les moelles sont isolés et recueillis sur la glace, réduire le volume de DPBS à un niveau où ils sont simplement recouverts de tampon. Utilisez une lame de rasoir stérile pour couper le moelles en cubes de 53 mm. </li><li> Fondre et arrondir l&#39;extrémité pointue d&#39;une pipette Pasteur stérile en maintenant brièvement dans une flamme. Assurez-vous que la pipette est refroidi avant de poursuivre. </li><li> Transférer la suspension de tissu à des tubes Falcon de 50 ml contenant une solution enzymatique stérile fraîchement préparée avec de la hyaluronidase et de la collagénase (voir 1.3.1) à l&#39;aide de la pipette Pasteur arrondie. Fermer le couvercle hermétiquement et incuber à 37 ° C sous agitation continue (220 rpm) dans un bain-marie de table ordinaire de 1,5 - 2 heures. </li><li> Par trituration de la suspension, une pipette le mélange de haut en bas plusieurs fois avec le tour pipette Pasteur (voir ci-dessus) jusqu&#39;à ce que la suspension devient homogène trouble. </li><li> Centrifuger la suspension pendant 5 min à 300 g et 16 ° C. Éliminer le surnageant, remettre le culot en détail dans DPBS, contenant de la gentamicine et la nystatine et centrifuger à nouveau. Répétez cette étape de lavage une fois. </li><li> Remettre en suspension les cellules dans un milieu entièrement complété et épépiner en cplats de ulture et / ou des plaques et micro-titrage. La préparation des deux médullaires (1 animale) donnera suffisamment de cellules pour une boîte de culture de 60 mm. </li><li> Après 24 h, se laver les cellules deux fois avec 600 mosmol DMEM et ajouter du milieu frais. Au cours de la semaine prochaine, les cellules doivent être lavés 2-3 fois (Figure 1). 6-8 jours après l&#39;ensemencement, les cellules IMCD peuvent être utilisés pour des expériences. N&#39;oubliez pas de les incuber dans un milieu sans DBcAMP et nystatine pendant 24 heures avant de commencer l&#39;expérience. Sinon AQP2 sera situé exclusivement dans la membrane plasmique. </li></ol>

<ol>
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Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Nous présentons un protocole détaillé pour la préparation et la mise en culture de cellules IMCD primaires de rat. L&#39;approche donne jusqu&#39;à 21 cm 2 de cellules provenant d&#39;un rat 20. L&#39;expérience nécessite un équipement de culture cellulaire standard et peut être réalisé par une seule personne au sein d&#39;environ 6 heures. Par conséquent, cette approche est appropriée comme une méthode de laboratoire standard. Cellules IMCD primaires de r...

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

Lors de la collecte des cellules rénales de conduits principaux, de l&#39;arginine-vasopressine (AVP) commande la réabsorption de l&#39;eau par la stimulation de l&#39;insertion de l&#39;eau canal aquaporine-2 (AQP2) dans la membrane plasmique. AVP se lie à la protéine de type à couplage de la vasopressine-2 récepteur du G (V2R) la stimulation de l&#39;adénylate cyclase et donc la formation de cAMP. L&#39;initiation de cette cascade de signalisation conduit à l&#39;activation de la protéine kinase A (PKA). PKA phosphoryle AQP2 à la sérine 256 (S256), qui est le déclencheur clé pour sa redistribution de vésicules intracellulaires dans la membrane plasmique. L&#39;insertion de la membrane facilite la réabsorption de l&#39;eau le long d&#39;un gradient osmotique et peaufine l&#39;homéostasie de l&#39;eau. Dysrégulation de la réabsorption de l&#39;eau AVP-dépendante, due à des aberrations de la sécrétion AVP ou les causes de signalisation AVP activés ou est associée à des maladies graves. Diminution de la réabsorption de l&#39;eau causée par des mutations du V2R ou AQP2 conduit à un diabète insipide néphrogénique, alors qu&#39;un AscATED plasma AVP niveau sanguin est associé à la réabsorption d&#39;eau excessive dans les maladies cardiovasculaires telles que l&#39;insuffisance cardiaque chronique et le syndrome de sécrétion inappropriée d&#39;hormone antidiurétique (SIADH). L&#39;importance de la réabsorption de l&#39;eau AVP-médiation découle non seulement de son implication dans la maladie. La translocation AVP-induite de vésicules AQP2 portant à la fusion et à la membrane plasmique représente un processus d&#39;exocytose strictement dépendante de l&#39;AMPc, qui n&#39;est actuellement pas bien compris. D&#39;autres exemples d&#39;une exocytose dépendante de l&#39;AMPc sont sécrétion de rénine dans le rein et H + sécrétion dans l&#39;estomac. Par conséquent, l&#39;élucidation des mécanismes moléculaires qui régissent le AQP2-translocation dans les cellules principales rénales permet non seulement de comprendre les processus moléculaires similaires dans d&#39;autres types de cellules, mais peut aussi ouvrir la voie à de nouvelles thérapies pour le traitement de maladies associées à des troubles de la réabsorption de l&#39;eau AVP-médiation . Elucidating mécanismes AQP2 contrôle nécessite la collecte des modèles cellulaires conduits principaux. Pour cela, un certain nombre de différentes lignées cellulaires de reins de mammifères sont disponibles. Cependant, ces modèles présentent plusieurs inconvénients. Dans de nombreux systèmes cellulaires niveau de la protéine de AQP2 est faible (tableau 1; M1, HEK293, COS-7, MDCK, LLC-PK1) 1-10, d&#39;autres ectopique surexpriment l&#39;homme (MCD4, WT10) 11,12 ou rat (CD8) 13 AQP2. En MCD4 et les cellules COS-7 du récepteur V2 n&#39;est pas exprimé. À notre connaissance, il n&#39;existe pas de ligne de la cellule permanente comme un modèle disponible, qui est dérivé du conduit rénal collecteur médullaire interne, cette partie du rein où AQP2 expression est la plus abondante, mais à partir du canal collecteur cortical 1,11,13-16 . Ces modèles cellulaires sont par exemple le immortalisé mpkCCD largement utilisé (par exemple 17) ou les 14 lignées cellulaires mTERT-CCD récemment établis. Les deux lignées cellulaires de façon endogène express AQP2 et V2R mais depuis ils sont issus de latube collecteur cortical contient probablement un protéome différent par rapport à collecteur médullaire interne les cellules des canaux (PCIME). Ils doivent par exemple exprimer différent Na + systèmes de transport. Ici, nous présentons un protocole pour la culture des cellules IMCD primaires exprimant V2R et AQP2 endogène. Ce modèle représente donc plus près la situation physiologique dans le tube collecteur rénal. En instituant la culture ne nécessite que étalons de laboratoire équipement d&#39;autres laboratoires peuvent facilement adopter cette approche.

<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

Arginine-vasopressine (AVP) facilite la réabsorption de l&#39;eau par une insuffisance rénale prélèvement de cellules principaux conduits et ainsi peaufine l&#39;homéostasie de l&#39;eau. AVP se lie à des récepteurs V2 de la vasopressine (V2R) sur la surface des cellules et induit ainsi la synthèse de l&#39;AMPc. Ceci stimule les processus de signalisation cellulaires conduisant à des changements dans la phosphorylation de la chaîne aquaporine-2 de l&#39;eau (AQP2). Protéine kinase A phoshorylates AQP2 et déclenche ainsi la translocation de AQP2 de vésicules intracellulaires dans la membrane plasmique de faciliter la réabsorption d&#39;eau à partir de l&#39;urine primaire. Aberrations de l&#39;AVP libération de la signalisation hypophyse ou AVP activé dans les cellules principales peuvent causer le diabète insipide central ou néphrogénique, respectivement, un taux sanguin AVP plasmatique élevé est associé aux maladies cardiovasculaires comme l&#39;insuffisance cardiaque chronique et le syndrome de sécrétion inappropriée d&#39;hormone antidiurétique. Ici, nous présentons un protocole pour cultivatisur des médullaire interne de rat primaire recueillir les cellules des canaux (PCIME), qui expriment V2R et AQP2 endogène. Les cellules sont appropriés pour l&#39;élucidation des mécanismes moléculaires qui sous-tendent le contrôle des AQP2 et ainsi de découvrir de nouvelles cibles thérapeutiques pour le traitement de maladies associées à une dérégulation de la réabsorption de l&#39;eau AVP-médiation. IMCD cellules sont obtenues à partir rat rénale moelles intérieure et sont utilisés pour des expériences de six à huit jours après le semis. IMCD cellules peuvent être cultivées dans des plats réguliers de culture cellulaire, des flacons et des plaques de micro-titrage de différents formats, la procédure ne nécessite que quelques heures, et est approprié pour les laboratoires de culture cellulaire standard.

La culture réussie des cellules IMCD primaires de rat se traduira par une monocouche confluente 6-8 jours après le semis (figure 2). Par boîte de 60 mm de la culture, il ya environ 6 x 10 6 cellules. Les cellules étroitement adhèrent aux boîtes de culture, car elles ont été recouvertes de collagène de type IV, un composant de la membrane basale 18. Par conséquent, les cellules IMCD ne se détache pas, même pendant plusieurs procédures approfondies de lavage. Jusqu&#39;?...

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

Arginine-vasopressine (AVP) contrôle fine-tuning de l&#39;homéostasie de l&#39;eau du corps en facilitant la réabsorption de l&#39;eau par les cellules rénales principales. Ici, nous présentons un protocole pour la culture de la médullaire interne de rat primaire recueillir les cellules des canaux appropriés pour l&#39;élucidation des mécanismes moléculaires qui sous-tendent la réabsorption de l&#39;eau AVP-médiation.

Culture primaire Rat collecteur médullaire interne les cellules des canaux

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 vues.  Max-Delbrück-Center for Molecular Medicine. Arginine-vasopressine (AVP) contrôle fine-tuning de l'homéostasie de l'eau du corps en facilitant la réabsorption de l'eau par les cellules rénales principales. Ici, nous présentons un protocole pour la culture de la médullaire interne de rat primaire recueillir les cellules des canaux appropriés pour l'élucidation des mécanismes moléculaires qui sous-tendent la réabsorption de l'eau AVP-médiation.

Vidéo: 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.

Protocole pour les neurones sympathiques de rat Culture ganglions cervicaux supérieur (SCG)

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

Recherche

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.

Culture primaire des adultes myocytes cœur de rat

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.

Primaire cellules épithéliales bronchiques humaines Cultivé à partir d&#39;explants

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.

Isolement de cellules basales et les cellules sous-muqueux du canal de la glande de la souris Trachée

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%.

L&#39;isolement et la culture primaire de cellules de foie de rat

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.

Isolement des cellules immunitaires des tumeurs primitives

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.

Culture de cellules humaines épithéliales nasales à l&#39;interface Air Liquide

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.

Un procédé de culture embryonnaire<em&gt; C. elegans</em&gt; Cellules

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-intérieur cellulaire massive Injection de cellules souches embryonnaires conduit à des taux plus élevés de chimérisme

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.

Culture primaire de cellules Corticosurrénaliennes Rat et tests des fonctions Stéroïdogéniques

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