Este trabajo fue apoyado por becas de Nacional de Salud y el Consejo de Investigación Médica de Australia (NHMRC) (526.615 y 545.906) y en parte por el Programa del Gobierno de Victoria OIS. DS es un compañero de NHMRC.

1. Preparación de [3 H]-colesterol <ol><li> En la campana de humos, dispensar la cantidad necesaria de colesterol [3 H] en un tubo de microcentrífuga de 1,5 ml (0,5 Ci (19 kBq) por pocillo se requiere para un ensayo típico). </li><li> Si [3 H]-colesterol se suspendió en tolueno, que se seque por completo con gas N2 y resuspender con 100% de etanol a una concentración final de 1 Ci (37 kBq / l. Vortex y se mezcla bien. </li></ol> 2. Células en placas y al etiquetado de colesterol celular Este protocolo ha sido probado con los siguientes tipos de células: monocitos humanos 4,5, THP-1 monocitos humanos macrófagos 6,7,8, RAW 264,7 macrófagos murinos 5,9,10,11, células HeLa 12, endoteliales de vena umbilical humana las células (HUVEC), las células BHK-21 9, fibroblastos humanos y de ratón, las células HepG2 13,14 hepatocarcinoma humano 15 y, en forma modificada, a partir de plaquetas &lt;sup&gt; 16. <ol><li> Resuspender las células y contar con ellos. Células de la placa en placas de 12 pocillos en la densidad final de 0.2x10 6 células por pocillo en 0,9 ml de medio completo. Las células seguirá creciendo y llegará a 0.8x10 6 células por pocillo en el momento del experimento de flujo de salida. Para las células que no se dividen, como diferenciadas células THP-1, la densidad de siembra debe ser 0.8x10 6 células por pocillo. Si se utiliza 6 - o placas de 24 pocillos, doble o la mitad del número de células por pocillo, respectivamente. El número de pozos debe ser suficiente para las determinaciones cuadruplicadas para cada condición experimental. </li><li> Si la diferenciación de células THP-1 se requiere, añadir PMA (concentración final 0,1 mg / ml) a los medios para 48-72 h. </li><li> Añadir una pequeña alícuota de los medios en el tubo de microcentrífuga que contiene la [3 H] colesterol. </li><li> Lave lados del tubo con los medios de comunicación antes de añadir [3H]-colesterol en una alícuota separada de medios completos (100 l / pocillo). </li> &lt;li&gt; Añadir los medios de comunicación que contienen [3 H] colesterol a los pozos con las células (volumen final por pocillo es de 1 ml). Si ningún tratamiento de las células se requiere antes de etiquetado, las células pueden ser cultivadas en [3 H] colesterol que contienen medio. </li><li> Se incuban las células durante 48 horas en cultivo celular incubadora (37 ° C, 5% de CO 2). </li></ol> 3. Equilibrio de incubación <ol><li> Después de la incubación de 48 horas, compruebe las células bajo el microscopio. Asegúrese de que estén sanos y se encuentran en aproximadamente el 80% de confluencia. </li><li> Retire los medios de comunicación que contiene [3 H] colesterol. Lave suavemente las células con PBS. Repita 3 veces el lavado. </li><li> Preparar medios libres de suero. Si es necesario, activar las células mediante la adición de agonistas de LXR (como A-901317 en 1.4 mmol / L) o cAMP (0,3 mmol / L; para las células de origen ratón) con el suero libre de los medios. </li><li> Añadir 500 l medios de comunicación libres de suero a cada pocillo. </li><li> Incubar durante 18 horas en la incubadora de cultivo celular (37 ° C, 5% de CO 2). </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 4. Incubación eflujo de colesterol <ol><li> Después de la incubación de 18 horas en medio libre de suero, comprobar las células bajo el microscopio. Asegúrese de que estén sanos y confluente (mínimo 80% de confluencia). </li><li> Preparar una solución de flujo de salida de colesterol aceptantes en medio libre de suero. Ejemplos de aceptadores incluyen: La apolipoproteína AI (ApoA-I) (concentración final de 10 mg / ml) Lipoproteína de alta densidad (HDL) (concentración final 20 ug / ml) Ciclodextrina (concentración final 200 mg / ml) Plasma (concentración final de 1-2%) </li><li> Lave suavemente las células con PBS. </li><li> Añadir 250 l de medio libre de suero con aceptores a cada pocillo. </li><li> Deja una serie de pozos para determinar el fondo de eflujo (flujo de salida de medio libre de suero con el NO aceptante) </li><li> Se incuban las células durante 2 horas en cultivo celular incubadora (37 ° C, 5% de CO 2). Duración de la incubación de eflujo puede variar desde 30 minutos a 8 horas si es necesario. </li></ol> 5. Procesamiento de Samples <ol><li> Después de 2 horas de incubación revisar las células bajo el microscopio. </li><li> Recoger los medios de comunicación en tubos de 1,5 ml de microfuga. Girar a 14.000 rpm durante 1-10 min a temperatura ambiente para eliminar los desechos celulares. </li><li> Transferir 100 l medios en 7 ml de vial de centelleo. Añadir 5 ml de Plus Insta-gel (PerkinElmer) y la mezcla de vórtice. Almacenar las muestras restantes a 4 ° C. </li><li> Colocar las placas en el congelador durante 30 minutos. Añadir 500 l DH 2 O a cada pocillo. Ver células bajo el microscopio para asegurar que todas las células han levantado desde el fondo de los pozos. Si todavía adheridas, o bien deje los platos con dH 2 O a 4 ° C durante la noche pozos o rasguño. </li><li> Una vez que todas las células ya han despegado, pipetee hacia arriba y hacia abajo para romper agregados celulares. Transferir 100 l en 7 ml viales de centelleo. Añadir 5 ml de Plus Insta-gel (PerkinElmer) y la mezcla de vórtice. Guarde el resto de las placas a 4 ° C. </li><li> Coloque viales de centelleo en un contador de centelleo. Configurar el contador para contar a [3H] endpm unidades. </li></ol> 6. El análisis de los resultados <ol><li> La tasa de eflujo de colesterol se expresa generalmente como una proporción de colesterol a partir de células movido al aceptor. La siguiente fórmula se utiliza: </li></ol><img alt="Ecuación 1" src="/files/ftp_upload/3810/3810eq1.jpg" /><ol start="2"><li> El flujo de salida específica se calcula como la diferencia entre el flujo de salida en la presencia o ausencia del aceptor (blanco). </li></ol><img alt="Ecuación 2" src="/files/ftp_upload/3810/3810eq2.jpg" /> 7. Línea de tiempo <img alt="Figura 1" src="/files/ftp_upload/3810/3810timeline.jpg" /> 8. Los resultados representativos Un ejemplo de un resultado de un experimento eflujo de colesterol se muestra en la fig. 1. En este experimento, THP-1 monocitos humanos se diferencian en macrófagos y el eflujo de colesterol a receptores diferentes eraprobado. Eflujo de colesterol y medio sin aceptantes (&quot;en blanco&quot;) fue de 0,79% y este valor fue considerado como un flujo de salida no son específicos y se restará de los demás valores. Eflujo de colesterol para la salud humana apoA-I (concentración final de 30 mg / ml) fue de 4,75%. Una muestra de plasma de referencia se incluyó en este experimento para controlar la variabilidad inter-experimental. Una muestra de referencia se puede utilizar para la normalización de datos a través de un gran número de experimentos, pero hemos encontrado prudente repetir el ensayo si la variabilidad es alta. Un plasma del paciente (concentración final 2%) se puso a prueba antes y después de que el paciente fue tratado con medicamentos. Se concluyó que en este medicamento paciente tenía un impacto negativo en la capacidad de plasma para apoyar el eflujo de colesterol. Otro ejemplo de un resultado del experimento eflujo se muestra en la fig. 2. En este experimento RAW 264.7 macrófagos fueron activados o no activados por incubación durante la noche con agonista LXR A-901317 (concentración final1 mol / L) y el eflujo de colesterol a la misma muestra de plasma humano (2%) se ensayó. Se concluyó que la activación de la expresión celular de los transportadores ABC con agonista LXR aumenta la capacidad de las células para liberar el colesterol a aceptor extracelular. <img alt="Figura 1" src="/files/ftp_upload/3810/3810fig1.jpg" /> Figura 1. Eflujo de colesterol de células THP-1 a aceptores diversos. Porcentaje de eflujo de colesterol (es decir, la proporción de colesterol marcado movido a partir de células al aceptor especificado) se muestra después de la sustracción de los valores en blanco. La media ± desviación estándar de las determinaciones de cuatro ejemplares, * p &lt;0,05. <img alt="Figura 2" src="/files/ftp_upload/3810/3810fig2.jpg" /> Figura 2. Eflujo de colesterol de RAW 264.7 activado o no activado con agonista LXR al plasma humano. Porcentaje de eflujo de colesterol (es decir, la proporción de colesterol marcada movido de células para elaceptor se especifica) se muestra después de la sustracción de los valores en blanco. La media ± desviación estándar de las determinaciones de cuatro ejemplares, * p &lt;0,001.

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Los autores declaran no tener ningún conflicto de intereses relacionados con este estudio.

El método descrito para medir el eflujo de colesterol está diseñado para medir el movimiento de colesterol de las células a un aceptor de colesterol extracelular. Hay varias consideraciones importantes en la comprensión de esta metodología. Etiquetado y el equilibrio En la metodología descrita colesterol marcado se añadieron al suero que contiene suero. Aunque nunca investigado en detalle, se supone que el colesterol se incorpora en las lipoproteínas de sue...

<table border="1"><tbody><tr><td> Reactivos \ células </td><td> HeLa </td><td> THP-1 </td><td> RAW </td><td> HUVEC </td><td> BHK-21 </td><td> HFF </td></tr><tr><td> Medios completos </td><td colspan="3"> RPMI - 10% de FCS - 1% de L-glutamina - Pen 0,2% por estreptococos </td><td> M199 - 5% de FCS - 1% de L-glutamina - Pluma de 1% por estreptococos - HEPES al 2% (1M) - 7% de las células endoteliales suplemento de crecimiento (ECG) </td><td colspan="2"> DMEM - 10% de FCS - 1% de L-glutamina - Pen 0,2% por estreptococos </td></tr><tr><td> Medio libre de suero </td><td colspan="3"> RPMI - 1% de L-glutamina - Pen 0,2% por estreptococos </td><td> M199 - 1% de L-glutamina - Pluma de 1% por estreptococos - HEPES al 2% (1M) - 7% de células endoteliales crecenº suplemento (ECG) </td><td colspan="2"> DMEM - 1% de L-glutamina - Pen 0,2% por estreptococos </td></tr><tr><td> Paso Método </td><td> 1 x Tripsina </td><td> Suspensión </td><td> Raspar </td><td colspan="3"> 1 x Tripsina </td></tr><tr><td> Paso Relación </td><td> 03:10 </td><td> 01:03 </td><td></td><td> 01:20 </td><td> 01:03 </td><td> 01:03 </td></tr><tr><td> Activación de las células (opcional) </td><td></td><td colspan="5"> A-901317 - LXR-agonista - Concentrado final. 4 micras, </td></tr><tr><td> Otros reactivos </td><td></td><td> forbol 12-miristato 13-acetato (PMA) - Diferenciación celular - Concentrado final. 0.1μg/ml </td><td></td><td></td><td></td><td></td></tr></tbody></table>

cholesterol efflux assay

El contenido de colesterol de las células deben mantenerse dentro de los límites muy estrechos, demasiado o demasiado poco de colesterol en unos resultados de células en la interrupción de las membranas celulares, la apoptosis y necrosis 1. Las células pueden colesterol fuente de la síntesis intracelular y de las lipoproteínas del plasma, ambas fuentes son suficientes para satisfacer plenamente las necesidades de células para el colesterol. Los procesos de la síntesis de colesterol y el consumo están fuertemente regulados y las deficiencias de colesterol son poco frecuentes 2. El exceso de colesterol es de 3 problema más común. Con la excepción de los hepatocitos y algunas células adrenocorticales grado, las células son incapaces de degradar el colesterol. Las células tienen dos opciones para reducir su contenido de colesterol: para convertir el colesterol en ésteres de colesterol, una opción con una capacidad limitada, como la sobrecarga de las células con ésteres de colesterol también es tóxico, y el eflujo de colesterol, una opción con la capacidad potencialmente ilimitada. Eflujo de colesterol es una especificaciónproceso de IFIC que está regulada por una serie de transportadores intracelulares, como el ATP vinculante cassette transportista proteínas A1 (ABCA1) y G1 (ABCG1) y B1 del receptor scavenger tipo. El aceptador natural de colesterol en plasma es de lipoproteína de alta densidad (HDL) y apolipoproteína AI. El ensayo de eflujo de colesterol está diseñado para cuantificar la tasa de eflujo de colesterol a partir de células cultivadas. Se mide la capacidad de las células para mantener el eflujo de colesterol y / o la capacidad de plasma aceptores para aceptar el colesterol liberado de las células. El ensayo consiste en los pasos siguientes. Paso 1: etiquetado colesterol celular mediante la adición de colesterol marcado a medio que contenía suero e incubando con células durante 24-48 h. Este paso puede ser combinado con la carga de las células con el colesterol. Paso 2: incubación de las células en medio libre de suero que se equilibre el colesterol marcado entre todos los grupos de colesterol intracelular. Esta etapa puede ser combinado con la activación de celular CHtransportistas olesterol. Paso 3: incubación de las células con aceptor extracelular y cuantificación de movimiento de colesterol marcado de las células al aceptor. Si los precursores de colesterol se utiliza para etiquetar el colesterol recién sintetizado, un cuarto paso, la purificación de colesterol, puede ser requerido. El ensayo proporciona la siguiente información: (i) cómo un tratamiento particular (una mutación, un derribo, la sobreexpresión de una o de un tratamiento) afecta a la capacidad de las células con el colesterol flujo de salida y (ii) cómo la capacidad de los aceptantes de plasma para aceptar el colesterol se ve afectada por una enfermedad o un tratamiento. Este método se utiliza a menudo en el contexto de la investigación cardiovascular, enfermedades metabólicas y neurodegenerativas, infecciosas y enfermedades reproductivas.

Watch this Scientific Journal Video about Cholesterol Efflux Assay at JoVE.com

Cholesterol Efflux Assay | Medicine | Video

Cholesterol Efflux Assay

El ensayo de colesterol está diseñado para cuantificar la tasa de eflujo de colesterol a partir de células cultivadas y la capacidad de plasma aceptores para aceptar el colesterol liberado de las células. El ensayo consiste en el etiquetado de las células con el colesterol, equilibración de colesterol entre piscinas intracelulares y la liberación de colesterol a un aceptor extracelular.

Ensayo de eflujo de colesterol

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

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SCIENTISTS IN ACTION

29.5K vistas.  Baker IDI Heart and Diabetes Institute. El ensayo de colesterol está diseñado para cuantificar la tasa de eflujo de colesterol a partir de células cultivadas y la capacidad de plasma aceptores para aceptar el colesterol liberado de las células. El ensayo consiste en el etiquetado de las células con el colesterol, equilibración de colesterol entre piscinas intracelulares y la liberación de colesterol a un aceptor extracelular.

Video: Cholesterol Efflux Assay

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Ensayo experimental de metástasis

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

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In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

Ensayo in vivo del canino función muscular

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

Un modelo de ratón de la Prueba de bolsillo para el Estudio de la angiogénesis de córnea

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

Spheroid Assay to Measure TGF-&amp;#946;-induced Invasion

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

Ensayo esferoide para medir TGF-β inducida por invasión

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

Un<em&gt; In vivo</em&gt; Ensayo de prueba de permeabilidad Vasos Sanguíneos

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

La<em&gt; En ovo</em&gt; CAM-ensayo como un Xenograftmodel para el sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

Simulación de páncreas Neuroplasticidad:<em&gt; In Vitro</em&gt; Dual-neurona Plasticidad Ensayo

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Tumor Ensayo tridimensional (3D) esferoide Invasion

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry. Image analyses of the cholesterol particles indicated that lovastatin, simvastatin, ezetimibe, and atorvastatin induce the formation of both globular and linear strand-shaped particles, whereas niacin, fibrates, fluvastatin, and rosuvastatin induce the formation of only globular-shaped particles. Next, purified very low-density lipoprotein (VLDL) and LDL particles incubated with these drugs showed changes in the morphology and image texture of cholesterol particles subpopulations. Furthermore, screening of 50 serum samples revealed the presence of a higher level of linear shaped HDL cholesterol particles in subjects with dyslipidemia (mean of 18.3%) compared to the age-matched normal (mean of 11.1%) samples. We also observed considerable variations in lipid-lowering drug effects on reducing linear shaped LDL and HDL cholesterol particles formation in serum samples. These findings indicate that lipid-lowering drugs, in addition to their cell-mediated hypolipidemic effects, may directly modulate morphology of cholesterol particles by a non-enzymatic mechanism of action. The outcomes of these results have potential to inform diagnosis of atherosclerosis and predict optimal lipid-lowering therapy.

The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

Efectos de fármacos hipolipemiantes en la modulación de la morfología de las partículas de colesterol

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.