Este trabajo fue apoyado por la Deutsche Forschungsgemeinschaft (DFG, KL y KL 1415/3-2 1415/4-2).

1. Preparación <ol><li> Preparación de las placas de cultivo <ol><li> Thaw colágeno tipo IV O / N a 4 ° C y se disuelven en 0,1% de ácido acético estéril. El volumen a utilizar depende del tipo y el número de platos en los que las células han de ser sembradas. Utilice 2 mg / cm 2. </li><li> Incubar los platos por lo menos durante 1 hora a TA y se lava dos veces con agua destilada (A. tridest). </li><li> Deje que los platos se sequen correctamente. </li></ol></li><li> La suplementación de medio <ol><li> Aumentar el nivel de glucosa de hasta 4,5 g / l mediante la adición de 1,75 g de glucosa a 500 ml de medio. Para ajustar el medio a 600 mOsmol, añadir NaCl 100 mM y urea 100 mM, para aumentar la osmolaridad de 200 mOsmol y 100 mOsmol, respectivamente. </li><li> Añadir ácidos 1% aminoácidos no esenciales, 1% Ultroser, 500 dbcAMP mM, 20 U / ml de nistatina y 0,25 g / ml de gentamicina (dilución 1:200 de solución madre con una concentración de 50 g / ml) a 600 mOsmol medio DMEM recién en el día de la preparación. </li></ol></li&gt; <li> Solución de la enzima <ol><li> Añadir 1 mg / ml de hialuronidasa y 2,2 mg / ml de colagenasa a DPBS que contiene 0,25 mg / ml de gentamicina y nistatina. Preparar 1 ml de esta solución de la enzima para la digestión de 2 riñón medullae interior. La solución debe ser esterilizado por filtración y se almacena en tubos Falcon de 50 ml. </li></ol></li></ol> 2. El cultivo de rata primaria medular interna recolección de células del conducto (IMCD) Todos los procedimientos de manipulación de los animales se llevarán a cabo de conformidad con la legislación local y federal. <ol><li> Anestesiar ratas en CO 2 (en una caja saturada con CO 2) o isoflurano (U-400 Unidad de Anestesia, Univentor Ltd., Malta, el flujo de aire 350 a 400 ml / min con 2-2,5% isoflurano). Decapita a los animales para el sangrado, que permite la detección exacta de la frontera entre el interior y el blanquecino médula externa más oscura. Además, el sangrado se reduce al mínimo el número de eritrocitos aislados. Retire los riñones ylavarlos en DPBS estéril suplementado con gentamicina y nistatina (véase más arriba) en hielo. Todos los pasos adicionales se llevarán a cabo en condiciones estériles banco limpio. </li><li> Transferir los riñones de la solución de DPBS en una compresa estéril para eliminar el exceso de líquido. Retire la cápsula del riñón graso con pinzas estériles y tijeras. </li><li> Habiendo el riñón todavía fijado en la compresa, cortado ligeramente al lado del eje longitudinal de la parte exterior (cortical). Asegúrese de no cortar por completo, pero dejarlo en una sola pieza, conectado en la pelvis renal. </li><li> Con tijeras curvas diseccionar cuidadosamente la medullae interior blanquecino, que están rodeados por la médula exterior brillante rosado. Recoger en DPBS que contiene gentamicina y nistatina en una placa de cultivo de 60 mm en el hielo. </li><li> Una vez que todos medullae se aíslan y se recogió en hielo, reducir el volumen de DPBS a un nivel en el que están cubiertos sólo con tampón. Use una hoja de afeitar estéril para cortar el medullae en cubos de 5mm 3. </li><li> Derretir y alrededor de la punta de una pipeta Pasteur estéril sosteniéndolo brevemente en una llama. Asegúrese de que la pipeta se enfría antes de continuar. </li><li> Transferir la suspensión de tejido a los tubos Falcon de 50 ml que contenían solución de enzima estéril recién preparado con hialuronidasa y colagenasa (véase 1.3.1) mediante el uso de la redondeada pipeta Pasteur. Cierre la tapa fuertemente y se incuba a 37 ° C bajo agitación continua (220 rpm) en un baño de agua de mesa regular para 1,5 - 2 horas. </li><li> Para la trituración de la suspensión, pipetear la mezcla arriba y abajo varias veces con la ronda pipeta Pasteur (véase más arriba) hasta que la suspensión turbia se vuelve homogénea. </li><li> Centrifugar la suspensión durante 5 min a 300 xg y 16 ° C. Descartar el sobrenadante, resuspender el precipitado a fondo en DPBS, que contiene gentamicina y nistatina y centrifugar de nuevo. Repita este paso de lavado una vez. </li><li> Resuspender las células en medio de ellos y de las semillas en c completamente suplementadoULTURA platos y / o placas de pocillos de microtitulación. La preparación de 2 medullae (1 animal) dará lugar a células suficientes para una placa de cultivo de 60 mm. </li><li> Después de 24 horas, lavar las células dos veces con 600 mosmol DMEM y añadir medio fresco. Durante la siguiente semana las células se deben lavar 2-3 veces (Figura 1). 6-8 días después de la siembra, las células IMCD se pueden utilizar para los experimentos. Recuerde que los incuban en medio sin dbcAMP y nistatina durante 24 horas antes de comenzar el experimento. De lo contrario AQP2 se encuentra exclusivamente en la membrana plasmática. </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

Se presenta un protocolo detallado para la preparación y el cultivo de células IMCD primarios de rata. El enfoque produce hasta 21 cm 2 de las células de una rata 20. El experimento requiere un equipo de cultivo celular estándar y puede ser llevada a cabo por una sola persona dentro de aproximadamente 6 horas. Por lo tanto, este enfoque es adecuado como un método estándar de laboratorio. Células IMCD primarios de rata se pueden sembrar en placas de cultivo de dif...

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

En la recogida de las células principales de conductos renales, arginina-vasopresina (AVP) controla la reabsorción de agua mediante la estimulación de la inserción del canal de agua acuaporina 2 (AQP2) en la membrana plasmática. AVP se une a la proteína de acoplamiento de la vasopresina de tipo receptor de G-2 (V2R) estimulación de la adenilato ciclasa y por lo tanto la formación de AMPc. La iniciación de esta cascada de señalización que conduce a la activación de la proteína quinasa A (PKA). PKA fosforila AQP2 en la serina 256 (S256), que es la clave de activación para su redistribución de las vesículas intracelulares a la membrana plasmática. La inserción en la membrana facilita la reabsorción de agua a lo largo de un gradiente osmótico y un ajuste fino de la homeostasis del agua corporal. La desregulación de la reabsorción de agua AVP-mediada, debido a las aberraciones de la secreción de AVP AVP o causas de señalización activadas por o se asocia con enfermedades graves. Disminución de la reabsorción de agua causada por mutaciones de V2R o AQP2 conduce a la diabetes insípida nefrogénica, mientras que un elevsangre ATED nivel AVP plasmática se asocia con la reabsorción de agua excesiva en las enfermedades cardiovasculares tales como la insuficiencia cardíaca crónica y el síndrome de secreción inapropiada de hormona antidiurética (SIADH). La importancia de la reabsorción de agua AVP mediada deriva no sólo de su implicación en la enfermedad. La translocación inducida por AVP de vesículas AQP2-cojinete a y fusión con la membrana plasmática representa un proceso exocítica estrictamente dependiente de cAMP, que actualmente no se entiende bien. Otros ejemplos para una exocitosis dependiente de AMPc son la secreción de renina en el riñón y la secreción de H + en el estómago. Por lo tanto, la elucidación de los mecanismos moleculares que regulan la AQP2-translocación en células renales principales no sólo ayuda a entender los procesos moleculares similares en otros tipos de células, pero también puede allanar el camino para nuevas terapias para el tratamiento de enfermedades asociadas con trastornos de la reabsorción de agua AVP mediada . Elucidating mecanismos AQP2 requiere el control de recogida de modelos celulares principales del conducto. Para esto un número de diferentes líneas celulares de riñón de mamífero están disponibles. Sin embargo, estos modelos tienen varios inconvenientes. En muchos sistemas de células el nivel de proteínas de AQP2 es baja (Tabla 1; M1, HEK293, COS-7, MDCK, LLC-PK1) 1-10, otros ectópica sobreexpresan humanos (MCD4, WT10) 11,12 o de rata (CD8) 13 AQP2. En MCD4 y células COS-7 no se expresa el receptor V2. Para nuestro conocimiento no hay una línea celular permanente como un modelo disponible, que se deriva de la renal interior conducto colector medular, que parte del riñón donde AQP2 expresión es más abundante, pero desde el conducto colector cortical 1,11,13-16 . Tales modelos celulares son por ejemplo el utilizado las mTERT-CCD de 14 líneas celulares establecidas recientemente mpkCCD inmortalizado (por ejemplo 17) o. Ambas líneas celulares expresan endógenamente AQP2 y V2R, pero ya que se derivan de lostúbulo colector cortical probablemente contiene un proteoma diferente en comparación con centro medular recoger células del conducto (IMCD). Por ejemplo, se deben expresar diferentes Na + los sistemas de transporte. A continuación, presentamos un protocolo de cultivo las células primarias que expresan IMCD V2R y AQP2 endógena. Este modelo, por lo tanto, representa más de cerca la situación fisiológica en el conducto colector renal. Como el establecimiento de la cultura requiere sólo equipo estándar de laboratorio otros laboratorios pueden adoptar fácilmente este método.

<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>
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			<td>Non essential amino acids (NEA)</td>
			<td>Biochrom AG</td>
			<td>K0293</td>
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			<td>Gentamicin</td>
			<td>Invitrogen GIBCO</td>
			<td>15710</td>
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			<td>DBcAMP</td>
			<td>BIOLOG</td>
			<td>D009</td>
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culturing primary rat inner medullary collecting duct cells

La arginina-vasopresina (AVP) facilita la reabsorción de agua por la recolección de células renales principales conductos y por lo tanto un ajuste fino de la homeostasis del agua corporal. AVP se une a los receptores de vasopresina V2 (V2R) sobre la superficie de las células y por lo tanto induce la síntesis de AMPc. Esto estimula los procesos de señalización celular que conducen a cambios en la fosforilación del canal de agua acuaporina 2 (AQP2). Proteína quinasa A phoshorylates AQP2 y de ese modo provoca la translocación de AQP2 de las vesículas intracelulares a la membrana plasmática facilitar la reabsorción de agua de la orina primaria. Las aberraciones de la liberación de AVP de la señalización de la pituitaria o AVP-activado en las células principales pueden causar diabetes insípida central o nefrogénica, respectivamente; un elevado nivel de sangre AVP plasmática se asocia con enfermedades cardiovasculares tales como la insuficiencia cardíaca crónica y el síndrome de secreción inapropiada de hormona antidiurética. A continuación, presentamos un protocolo para cultivatiel de la rata medular interna primaria recoger células del conducto (IMCD), que expresan V2R y AQP2 endógenamente. Las células son adecuados para elucidar los mecanismos moleculares que subyacen en el control de AQP2 y por lo tanto para descubrir nuevas dianas farmacológicas para el tratamiento de enfermedades asociadas con la disregulación de la reabsorción de agua AVP mediada. IMCD células se obtienen a partir de rata renal medullae interior y se utilizan para los experimentos de seis a ocho días después de la siembra. IMCD células pueden ser cultivadas en platos de cultivo de células regulares, matraces y placas de microtitulación de diferentes formatos, el procedimiento sólo requiere unas pocas horas, y es apropiado para laboratorios de cultivo celular estándar.

El cultivo exitoso de células IMCD primarios de rata se traducirá en una monocapa confluente 6-8 días después de la siembra (Figura 2). Per 60 mm placa de cultivo hay aproximadamente 6 x 10 6 células. Las células se adhieren firmemente a las placas de cultivo, ya que estos fueron recubiertas con colágeno tipo IV, un componente de la membrana basal 18. Por lo tanto, las células IMCD no separar incluso durante varios procedimientos de lavado a fondo. Hasta el 80% de las célu...

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

La arginina-vasopresina (AVP) controla la puesta a punto de la homeostasis del agua corporal facilitando la reabsorción de agua por las células principales renales. A continuación, se presenta un protocolo para el cultivo de la rata medular interna primaria recoger células de los conductos adecuados para la elucidación de los mecanismos moleculares que subyacen a la reabsorción de agua AVP-mediada.

El cultivo primarios de rata medular interna recolección de células del conducto

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 vistas.  Max-Delbrück-Center for Molecular Medicine. La arginina-vasopresina (AVP) controla la puesta a punto de la homeostasis del agua corporal facilitando la reabsorción de agua por las células principales renales. A continuación, se presenta un protocolo para el cultivo de la rata medular interna primaria recoger células de los conductos adecuados para la elucidación de los mecanismos moleculares que subyacen a la reabsorción de agua AVP-mediada.

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.

Protocolo para las neuronas simpáticas de rata cultivo Ganglios cervical superior (SCG)

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

Investigación

Biología

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.

Cultivo primario de miocitos adultos de corazón de rata

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.

Primaria células epiteliales bronquiales Cultivada a partir de explantes

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.

Aislamiento de células basales y de células submucosas conducto de la glándula de ratón tráquea

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%.

Aislamiento y cultivo primario de células hepáticas de ratas

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.

El aislamiento de las células inmunes de tumores primarios

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.

El cultivo de células humanas epiteliales nasales en la interfase aire líquido

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 método para cultivar embrionarias<em&gt; C. elegans</em&gt; Células

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-interno celular masiva inyección de células madre embrionarias conduce a mayores tasas de quimerismo

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.

Cultivo primario de células adrenocorticales rata y análisis de funciones Steroidogenic

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