Questo lavoro è stato sostenuto dalla Deutsche Forschungsgemeinschaft (DFG; KL KL 1415/3-2 e 1415/4-2).

1. Preparazione <ol><li> Preparare i piatti della cultura <ol><li> Disgelo collagene tipo IV O / N a 4 ° C e sciogliere in 0,1% di acido acetico sterile. Il volume da utilizzare dipende dal tipo e dal numero di piatti in cui le cellule devono essere seminato. Usare 2 ug / cm 2. </li><li> Incubare piatti almeno per 1 ora a RT e lavare due volte con acqua distillata (A. tridest). </li><li> Lasciare i piatti ad asciugare correttamente. </li></ol></li><li> La supplementazione di medie <ol><li> Aumentare il livello di glucosio fino a 4,5 g / L aggiungendo 1,75 g di glucosio a 500 ml di terreno. Per regolare il mezzo a 600 mosmol, aggiungere 100 mM NaCl 100 mM e urea, per aumentare l&#39;osmolarità da 200 mosmol e 100 mosmol, rispettivamente. </li><li> Aggiungere 1% di acidi non essenziali aminoacidi, 1% ultroser, 500 pM dbcAMP, 20 U / ml nistatina e 0,25 mcg / ml gentamicina (diluizione 1:200 di soluzione madre con una concentrazione di 50 ug / ml) a 600 mosmol mezzo DMEM fresco il giorno della preparazione. </li></ol></li&gt; <li> Soluzione enzimatica <ol><li> Aggiungere 1 mg / ml di ialuronidasi e 2,2 mg / ml di collagenasi di DPBS-contenente 0,25 mg / ml di gentamicina e nistatina. Preparare 1 ml di questa soluzione di enzimi per la digestione di 2 rene midollare interna. La soluzione deve essere sterilizzata per filtrazione ed è memorizzato in 50 ml provette Falcon. </li></ol></li></ol> 2. La coltivazione di primaria Rat midollare interna dotto collettore (IMCD) Cells Tutte le procedure di movimentazione animali devono essere effettuati in conformità con la legislazione locale e federale. <ol><li> Anestetizzare topi in CO 2 (in una scatola saturo di CO 2) o isoflurano (U-400 Anestesia unità, Univentor Ltd., Malta, il flusso d&#39;aria 350-400 ml / min con 2-2.5% isoflurano). Decapitare gli animali per i sanguinamenti che consente il rilevamento esatto del confine tra il biancastro interiore e la midollare esterna più scura. Inoltre, il sanguinamento minimizza il numero di eritrociti isolati. Rimuovere i reni elavarli in DPBS sterile addizionato con gentamicina e nistatina (vedi sopra) in ghiaccio. Gli ulteriori passi devono essere effettuate in condizioni sterili banco pulito. </li><li> Trasferire i reni dalla soluzione DPBS su una compressa sterile per rimuovere il liquido in eccesso. Rimuovere la capsula renale grasso con pinze e forbici sterili. </li><li> Avendo il rene ancora risolto nel comprimere, taglio leggermente vicino all&#39;asse longitudinale del (corticale) lato esterno. Assicurarsi di non tagliare completamente, ma di lasciarla in un unico pezzo, collegato nella pelvi renale. </li><li> Con forbici curve con attenzione sezionare la midollare interna biancastra, che sono circondati dal luccicante midollare esterna rosato. Li Raccogliere in DPBS, contenente gentamicina e nistatina in una capsula di Petri 60 millimetri sul ghiaccio. </li><li> Una volta che tutti midollare sono isolati e raccolti su ghiaccio, ridurre il volume di DPBS ad un livello in cui essi sono solo coperti con tampone. Utilizzare una lametta sterile per tagliare la midollare a cubetti di 53 mm. </li><li> Fondere e arrotondare la punta di una pipetta Pasteur sterile tenendolo per breve tempo in una fiamma. Assicurarsi che la pipetta viene raffreddata prima di continuare. </li><li> Trasferire la sospensione dei tessuti per i 50 ml provette Falcon contenenti preparati al momento soluzione enzimatica sterile con ialuronidasi e collagenasi (vedi 1.3.1) utilizzando il arrotondata pipetta Pasteur. Chiudere il coperchio e incubare a 37 ° C sotto continua agitazione (220 rpm) a bagnomaria tavolo regolare per 1,5 - 2 ore. </li><li> Per triturazione della sospensione, la miscela pipettare su e giù diverse volte al tondo pipetta Pasteur (vedere sopra) finché la sospensione diventa torbida omogenea. </li><li> Centrifugare la sospensione per 5 minuti a 300 xg e 16 ° C. Eliminare il surnatante, risospendere il pellet a fondo in DPBS, contenente gentamicina e nistatina e centrifugare di nuovo. Ripetere questa fase di lavaggio una volta. </li><li> Risospendere le cellule in terreno completamente integrato e loro seme in cpiatti ultura e / o piastre e micro-titolazione. La preparazione di 2 midollare (1 animali) produce cellule sufficiente per un 60 millimetri piatto di coltura. </li><li> Dopo 24 ore, lavare le cellule due volte con 600 mosmol DMEM e aggiungere mezzo fresco. Durante la settimana successiva le cellule devono essere lavati 2-3 volte (Figura 1). 6-8 giorni dopo la semina, le cellule IMCD possono essere usate per esperimenti. Ricordate di incubare loro in mezzo senza dbcAMP e nistatina per 24 ore prima di iniziare l&#39;esperimento. Altrimenti AQP2 sarà situato esclusivamente nella membrana plasmatica. </li></ol>

<ol>
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Gli autori dichiarano di non avere interessi finanziari in competizione.

Vi presentiamo un protocollo dettagliato per la preparazione e la coltura di cellule IMCD primari di ratto. L&#39;approccio produce fino a 21 cm 2 di cellule da un ratto 20. L&#39;esperimento richiede apparecchiatura standard coltura cellulare e può essere effettuata da una sola persona entro circa 6 ore. Pertanto, questo approccio è adatto come metodo di laboratorio standard. Cellule IMCD primari di ratto possono essere seminate in piastre di coltura di diverse dimen...

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

Nella raccolta di cellule principali del dotto renali, arginina-vasopressina (AVP) controlla il riassorbimento dell&#39;acqua stimolando l&#39;inserimento del canale dell&#39;acqua aquaporina-2 (AQP2) nella membrana plasmatica. AVP si lega alla proteina-accoppiato vasopressina di tipo-2 recettore G (V2R) stimolare ciclasi e quindi formazione di cAMP. Apertura di questa cascata di segnalazione porta all&#39;attivazione della proteina chinasi A (PKA). PKA fosforila AQP2 a serina 256 (S256), che è la chiave di innesco per la sua ridistribuzione da vescicole intracellulari nella membrana plasmatica. L&#39;inserimento della membrana facilita il riassorbimento dell&#39;acqua lungo un gradiente osmotico e di una messa a punto del corpo omeostasi acqua. Disregolazione del riassorbimento dell&#39;acqua AVP-mediata, a causa della secrezione di AVP aberrazioni o cause di segnalazione AVP-attivati ​​o è associata a malattie gravi. Diminuzione del riassorbimento di acqua causata da mutazioni di V2R o AQP2 porta a diabete insipido nefrogenico, mentre un elevated livello di AVP plasma sanguigno è associato a un eccessivo riassorbimento di acqua a malattie cardiovascolari come l&#39;insufficienza cardiaca cronica e la sindrome da inappropriata secrezione di ormone antidiuretico (SIADH). Il significato di riassorbimento dell&#39;acqua AVP-mediata deriva non solo dalla sua implicazione nella malattia. La traslocazione AVP-indotta di vescicole AQP2-cuscinetto a e la fusione con la membrana plasmatica rappresenta un processo di esocitosi strettamente cAMP-dipendente, che attualmente non è ben compreso. Altri esempi per una esocitosi cAMP-dipendente sono secrezione di renina nel rene e H + secrezione nello stomaco. Pertanto, delucidazione dei meccanismi molecolari che regolano la traslocazione-AQP2 in cellule renali principali non solo aiuta a comprendere i processi molecolari simili in altri tipi cellulari, ma può anche aprire la strada a nuove terapie per il trattamento di malattie associate a disturbi del riassorbimento dell&#39;acqua AVP-mediata . Elucidating meccanismi AQP2 controllo richiede la raccolta modelli di cellule principali del dotto. Per questo un certo numero di differenti linee cellulari di rene di mammifero sono disponibili. Tuttavia, questi modelli hanno diversi inconvenienti. In molti sistemi di celle a livello della proteina di AQP2 è basso (Tabella 1; M1, HEK293, COS-7, MDCK, LLC-PK1) 1-10, altri ectopicamente iperesprimono umani (MCD4, WT10) 11,12 o ratto (CD8) 13 AQP2. In MCD4 e cellule COS-7 il recettore V2 non viene espresso. A nostra conoscenza non vi è alcuna linea cellulare permanente come un modello disponibile, che è derivato dal renale midollare interna dotto collettore, la parte del rene AQP2 cui espressione è più abbondante, ma dal dotto collettore corticale 1,11,13-16 . Tali modelli cellulari sono per esempio il diffuso immortalato i mTERT-CCD 14 linee cellulari di recente istituzione mpkCCD (ad esempio 17) o. Entrambe le linee cellulari esprimono endogenamente AQP2 e V2R, ma dal momento che sono derivati ​​daldotto collettore corticale molto probabilmente contiene un proteoma diverso rispetto a midollare interna dotto collettore (IMCD) cellule. Devono per esempio esprimere diversa Na + sistemi di trasporto. Qui vi presentiamo un protocollo per colture primarie cellule IMCD esprimono V2R e AQP2 endogeno. Questo modello, quindi, rappresenta più fedelmente la situazione fisiologica nel dotto collettore renale. Come istituisce la cultura richiede solo normali attrezzature di laboratorio altri laboratori possono facilmente adottare questo approccio.

<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

Arginina-vasopressina (AVP) facilita il riassorbimento di acqua da parte renale raccogliendo cellule principali del dotto e quindi una messa a punto del corpo omeostasi acqua. AVP si lega ai recettori V2 della vasopressina (V2R) sulla superficie delle cellule e induce quindi la sintesi di cAMP. Questo stimola processi di segnalazione cellulari che portano ai cambiamenti nella fosforilazione del canale dell&#39;acqua aquaporina-2 (AQP2). Proteina chinasi A phoshorylates AQP2 e innesca così la traslocazione di AQP2 da vescicole intracellulari nella membrana plasmatica facilitando riassorbimento di acqua da urina primaria. Aberrazioni di AVP liberazione dalla segnalazione ipofisi o AVP-attivato nelle cellule principali possono causare il diabete insipido centrale o nefrogenica, rispettivamente, un plasma sanguigno livello AVP elevata è associata a malattie cardiovascolari come l&#39;insufficienza cardiaca cronica e la sindrome da inappropriata secrezione di ormone antidiuretico. Qui vi presentiamo un protocollo per cultivatiil ratto di primaria midollare interna dotto collettore (IMCD) cellule, che esprimono V2R e AQP2 endogena. Le cellule sono adatti per chiarire i meccanismi molecolari alla base del controllo di AQP2 e quindi di scoprire nuovi bersagli farmacologici per il trattamento di malattie associate alla disregolazione del riassorbimento dell&#39;acqua AVP-mediata. Cellule IMCD sono ottenuti da topo renale midollare interna e sono utilizzati per esperimenti da sei a otto giorni dopo la semina. Cellule IMCD possono essere coltivate in piatti normali di coltura cellulare, boccette e piastre micro-titolo di formati diversi, la procedura richiede solo poche ore, ed è adatto per laboratori standard di coltura cellulare.

La coltivazione di successo di cellule IMCD primari di ratto si tradurrà in un monostrato confluente 6-8 giorni dopo la semina (Figura 2). A 60 millimetri piatto di coltura sono circa 6 x 10 6 cellule. Le cellule strettamente aderire alle piastre di coltura, in quanto questi sono stati rivestiti con collagene di tipo IV, un componente membrana basale 18. Pertanto, le cellule IMCD non staccarsi nemmeno durante le diverse procedure di lavaggio accurato. Fino al 80% delle cellule in ...

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

Arginina-vasopressina (AVP) controlla la messa a punto del corpo omeostasi acqua facilitando il riassorbimento di acqua da parte delle cellule principali renali. Qui vi presentiamo un protocollo per la coltivazione di ratto primario midollare interna raccogliere cellule del dotto adatti per la delucidazione dei meccanismi molecolari alla base di riassorbimento di acqua AVP-mediata.

Coltura primaria Rat midollare interna Raccolta cellule del dotto

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 Views.  Max-Delbrück-Center for Molecular Medicine. Arginina-vasopressina (AVP) controlla la messa a punto del corpo omeostasi acqua facilitando il riassorbimento di acqua da parte delle cellule principali renali. Qui vi presentiamo un protocollo per la coltivazione di ratto primario midollare interna raccogliere cellule del dotto adatti per la delucidazione dei meccanismi molecolari alla base di riassorbimento di acqua AVP-mediata.

Video: 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.

Protocollo per la coltura neuroni simpatico da Rat gangli cervicale superiore (SCG)

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

Ricerca

Biologia

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.

Cultura primario di miociti adulti Cuore 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.

Primario cellule epiteliali bronchiali umane Grown da espianti

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.

Isolamento delle cellule basali e sottomucosa cellule del dotto di Gland 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. ...

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%.

Isolamento e la coltura primaria di cellule epatiche di ratto

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.

Isolamento di cellule immunitarie di tumori primari

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.

Coltura di cellule umane nasale epiteliali presso l&#39;Air Interface Liquid

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 metodo per la coltura embrionale<em&gt; C. elegans</em&gt; Celle

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-inner Cell Mass iniezione delle cellule staminali embrionali porta a tassi più elevati di chimerismo

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.

Colture primarie di cellule corticosurrenali ratto e saggi di Steroidogenic funzioni

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