Este trabalho foi suportado por concessões do National Saúde e Pesquisa Médica do Conselho da Austrália (NHMRC) (526.615 e 545.906) e em parte pelo Programa do Governo de Victoria da OIS. DS é um companheiro de NHMRC.

1. Preparação de [3 H]-colesterol <ol><li> Na hotte, dispensar a quantidade necessária de [3 H] colesterol em um tubo de microcentrífuga 1,5 ml (0,5 uCi (19 kBq) por poço é necessário para um ensaio típico). </li><li> Se [3 H]-colesterol é suspenso em tolueno, secá-la para baixo completamente com N2 gás e ressuspender com etanol a 100% para uma concentração final de 1 uCi (37 kBq / uL Vortex. E ​​misturar bem. </li></ol> 2. Células proteções e Rotulagem colesterol celular Este protocolo foi testado utilizando os seguintes tipos de células: monócitos humanos 4,5, THP-1 humanos monócitos-macrófagos 6,7,8, RAW 264.7 macrófagos murinos 5,9,10,11, células HeLa 12, de veia umbilical humana endotelial As células (HUVEC) e células BHK-21 9, fibroblastos humanos e rato 13,14, células HepG2 humanas hepatocarcinoma 15 e, na forma modificada, a partir de plaquetas &lt;sup&gt; 16. <ol><li> Ressuspender as células e contá-las. Células da placa em 12 poços na densidade final de 0.2x10 6 células por poço em 0,9 ml de meio completo. Células irá continuar a crescer e atingirá 0.8x10 6 células por poço por o tempo da experiência de efluxo. Para as células que não se dividem, tais como diferenciadas células THP-1, a densidade de sementeira deve ser 0.8x10 6 células por poço. Se usando 6 - ou placas de 24 poços, duplo ou metade do número de células por poço, respectivamente. O número de poços deve ser suficiente para determinações quadruplicado para cada condição experimental. </li><li> Se a diferenciação de células THP-1 é necessária, adicionar PMA (concentração final de 0,1 ug / ml) aos meios de comunicação para 48-72 h. </li><li> Adicionar uma pequena alíquota de meios para dentro do tubo de microcentrífuga contendo o [3 H] colesterol. </li><li> Lavar os lados do tubo com os meios de antes da adição de [3 H] colesterol em uma alíquota separada de meio completo (100 uL / poço). </li> &lt;li&gt; Adicionar os meios contendo [3 H] colesterol para os poços com células (volume final por alvéolo é de 1 ml). Se nenhum tratamento de células é necessária antes de rotulagem, as células podem ser colocadas em [H 3] meio contendo colesterol. </li><li> Incubar as células durante 48 horas em cultura de células incubadora (37 ° C CO, 5% 2). </li></ol> 3. Incubação de equilíbrio <ol><li> Após 48 horas de incubação, verificar células ao microscópio. Garantir que eles são saudáveis ​​e estão em confluência de aproximadamente 80%. </li><li> Remova a mídia contendo [3 H] colesterol. Lave as células cuidadosamente com PBS. Repita 3 vezes de lavagem. </li><li> Preparar meios isentos de soro. Se necessário, ativar as células adicionando agonista LXR (como TO-901317 a 1-4 mMol / L) ou AMPc (0,3 mmol / L; para células de origem do mouse apenas) para soro livre-media. </li><li> Adicionar 500 ul de soro meios de comunicação livres para cada poço. </li><li> Incubar durante 18 horas na célula incubadora de cultura (37 ° C CO, 5% 2). </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 4. Incubação efluxo do colesterol <ol><li> Após 18 horas de incubação em meio isento de soro, vá células ao microscópio. Garantir que eles são saudáveis ​​e confluente (mínimo de 80% de confluência). </li><li> Preparar uma solução de colesterol aceitadores de efluxo em meio isento de soro. Exemplos de aceitadores incluem: A apolipoproteína AI (apo AI) (concentração final 10 ug / ml) Lipoproteína de alta densidade (HDL) (concentração final 20 ug / ml) Ciclodextrina (concentração final 200 ug / ml) Plasma (concentração final de 1-2%) </li><li> Lave as células cuidadosamente com PBS. </li><li> Adicionar 250 uL de meios isentos de soro com aceitadores a cada poço. </li><li> Deixar um conjunto de poços para determinar fundo de efluxo (efluxo de mídia livre com soro NO aceitante) </li><li> Incubar as células durante 2 horas em cultura de células incubadora (37 ° C, 5% de CO 2). Duração da incubação pode variar de efluxo 30 min a 8 h, se necessário. </li></ol> 5. Processamento Samples <ol><li> Após 2 horas de incubação verificar células ao microscópio. </li><li> Colete mídia em tubos de microcentrífuga de 1,5 ml. Girar a 14.000 rpm para min 1-10 à temperatura ambiente para remover os restos celulares. </li><li> Transferir 100 uL meios em 7 ml frasco de cintilação. Adicionar 5 ml de gel de Insta-Plus (PerkinElmer) e mistura de vórtice. Armazenar amostras restantes a 4 ° C. </li><li> Colocar as placas no congelador durante 30 minutos. Adicionar 500 uL de dH2O a cada poço. Verificar as células sob microscópio para assegurar que todas as células têm levantada a partir do fundo dos poços. Se ainda aderente, deixe placas com dH 2 O a 4 ° C poços durante a noite ou arranhão. </li><li> Uma vez que todas as células têm levantado, pipetar cima e para baixo para quebrar aglomerados celulares. Transferir 100 uL em frascos de cintilação 7 ml. Adicionar 5 ml de gel de Insta-Plus (PerkinElmer) e mistura de vórtice. Armazenar placas restantes a 4 ° C. </li><li> Coloque frascos de cintilação em contador de cintilação. Configurar contador para contar para [3 H] emunidades DPM. </li></ol> 6. Analisando os resultados <ol><li> A taxa de efluxo de colesterol é geralmente expressa como uma proporção de colesterol movido a partir de células para o aceitador. A fórmula seguinte é utilizado: </li></ol><img alt="Equação 1" src="/files/ftp_upload/3810/3810eq1.jpg" /><ol start="2"><li> O efluxo específica é calculada como a diferença entre o efluxo na presença ou ausência do aceitador (branco). </li></ol><img alt="Equação 2" src="/files/ftp_upload/3810/3810eq2.jpg" /> 7. Linha do tempo <img alt="A Figura 1" src="/files/ftp_upload/3810/3810timeline.jpg" /> 8. Os resultados representativos Um exemplo de um resultado de uma experiência de efluxo de colesterol é mostrado na fig. 1. Nesta experiência THP-1 monócitos humanos foram diferenciadas em macrófagos e de efluxo de colesterol para aceitadores diferentes foitestado. Efluxo de colesterol e médio sem receptores (&quot;em branco&quot;) foi de 0,79% e este valor foi considerado como um efluxo não específico e foi subtraído de outros valores. Efluxo de colesterol para a saúde humana apoA-I (concentração final 30 ug / ml) foi de 4,75%. Uma amostra de plasma de referência foram incluídos nesta experiência para controlar inter-experimentais variabilidade. Uma amostra de referência pode ser utilizado para a normalização de dados através de um grande número de experiências, mas que encontramos prudente repetir o ensaio se a variabilidade é alta. Um plasma do paciente (concentração final de 2%) foi testada antes e depois de o paciente foi tratado com a medicação. Concluiu-se que neste medicação do paciente teve um impacto negativo sobre a capacidade do plasma para suportar o efluxo de colesterol. Outro exemplo de um resultado da experiência de efluxo é mostrado na fig. 2. Neste experimento RAW 264.7 de macrófagos foram ativados ou não ativados por incubação durante a noite com LXR agonista TO-901317 (concentração final1 mmol / L) e de efluxo de colesterol para a mesma amostra de plasma humano (2%) foi testado. Concluiu-se que a activação da expressão celular de transportadores ABC com agonista LXR aumenta a capacidade das células para libertar o colesterol para o aceitador extracelular. <img alt="A Figura 1" src="/files/ftp_upload/3810/3810fig1.jpg" /> Figura 1. Efluxo de colesterol a partir de células THP-1 a receptores de vários. Percentagem de efluxo de colesterol (isto é, a proporção de colesterol marcado movido a partir de células para o aceitador especificado) é mostrada após subtracção dos valores em branco. Média ± DP de determinações quadruplicado, * p &lt;0,05. <img alt="A Figura 2" src="/files/ftp_upload/3810/3810fig2.jpg" /> Figura 2. Efluxo de colesterol a partir de 264,7 Percentagem activado ou não activado com LXR agonista ao plasma humano. RAW de efluxo de colesterol (isto é, a proporção de colesterol marcado movido a partir de células àaceitador especificado) é mostrada após subtracção dos valores em branco. Média ± DP de determinações quadruplicado, * p &lt;0,001.

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Os autores declaram sem conflitos de interesses relacionados a este estudo.

O método descrito para medir o efluxo de colesterol é concebido para medir o movimento do colesterol a partir de células de um aceitador de colesterol extracelular. Há várias considerações críticas na compreensão desta metodologia. Rotulagem e de equilíbrio Na metodologia descrita colesterol marcado é adicionado ao soro contendo soro. Embora nunca investigada em detalhe, presume-se que o colesterol é incorporado nas lipoproteínas do soro e são absorvid...

<table border="1"><tbody><tr><td> Reagentes \ 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> De mídia completo </td><td colspan="3"> RPMI - FCS 10% - 1% de L-glutamina - Strep Pen 0,2% </td><td> M199 - FCS 5% - 1% de L-glutamina - Strep Pen 1% - HEPES 2% (1M) - 7% suplemento de crescimento de células endoteliais (ECGS) </td><td colspan="2"> DMEM - FCS 10% - 1% de L-glutamina - Strep Pen 0,2% </td></tr><tr><td> Séricos Mídia Livre </td><td colspan="3"> RPMI - 1% de L-glutamina - Strep Pen 0,2% </td><td> M199 - 1% de L-glutamina - Strep Pen 1% - HEPES 2% (1M) - Célula endotelial crescer 7%º suplemento (ECGS) </td><td colspan="2"> DMEM - 1% de L-glutamina - Strep Pen 0,2% </td></tr><tr><td> Método passagem </td><td> 1 x Tripsina </td><td> Suspensão </td><td> Raspar </td><td colspan="3"> 1 x Tripsina </td></tr><tr><td> Relação de passagem </td><td> 3:10 </td><td> 01:03 </td><td></td><td> 1:20 </td><td> 01:03 </td><td> 01:03 </td></tr><tr><td> Ativação de células (opcional) </td><td></td><td colspan="5"> TO-901.317 - LXR-agonista - Conc final. 4 mm </td></tr><tr><td> Outros reagentes </td><td></td><td> forbol 12-miristato 13-acetato (PMA) - Diferenciação celular - Conc final. 0.1μg/ml </td><td></td><td></td><td></td><td></td></tr></tbody></table>

cholesterol efflux assay

Teor de colesterol de células deve ser mantida dentro dos limites muito apertados, muito ou pouco colesterol em alguns resultados de células em ruptura das membranas celulares, a apoptose e necrose 1. As células podem colesterol fonte da síntese intracelular e de lipoproteínas plasmáticas, ambas as fontes são suficientes para satisfazer plenamente as necessidades de células para o colesterol. Os processos de síntese de colesterol e absorção são fortemente regulados e deficiências de colesterol são 2 raro. Excesso de colesterol é de 3 problema mais comum. Com a excepção dos hepatócitos e para algumas células grau adrenocorticais, as células são incapazes de degradar o colesterol. As células têm duas opções para reduzir seu teor de colesterol: para converter colesterol em ésteres de colesterol, uma opção com capacidade limitada como sobrecarga células com ésteres de colesterol também é tóxico, e efluxo do colesterol, uma opção com capacidade potencialmente ilimitado. Efluxo do colesterol é uma especificaçãoprocesso ific que é regulada por uma série de transportadores intracelulares, tais como ligação de ATP cassete transportador proteínas A1 (ABCA1) e G1 (ABCG1) e B1 eliminador de tipo de receptor. O aceitador de natural de colesterol no plasma é de lipoproteína de alta densidade (HDL) e da apolipoproteína AI. O ensaio de efluxo de colesterol é concebido para quantificar a taxa de efluxo de colesterol a partir de células cultivadas. Mede a capacidade das células para manter o efluxo de colesterol e / ou a capacidade de plasma aceitadores para aceitar o colesterol libertado a partir de células. O ensaio consiste nos passos seguintes. Passo 1: etiquetagem colesterol celular por adição de colesterol rotulados para meio contendo soro e incubando com células de 24-48 h. Este passo pode ser combinado com o carregamento de células com colesterol. Passo 2: a incubação das células em meio isento de soro para equilibrar o colesterol marcado entre todos os pools de colesterol intracelular. Esta fase pode ser combinado com a activação do celular CHtransportadores olesterol. Passo 3: a incubação de células com aceitador extracelular e quantificação de movimento do colesterol a partir de células marcadas para o aceitador. Se precursores de colesterol foram utilizados para etiquetar o colesterol recém-sintetizado, um quarto passo, a purificação do colesterol, pode ser necessária. O ensaio proporciona a seguinte informação: (i) como um tratamento particular (uma mutação, um knock-baixo, a superexpressão um ou um tratamento) afecta a capacidade da célula para o colesterol de efluxo e (ii) como a capacidade de aceitadores de plasma para aceitar o colesterol é afectada por uma doença ou um tratamento. Este método é geralmente usado no contexto de pesquisas cardiovasculares, doenças metabólicas e neurodegenerativas, infecciosas e doenças reprodutivas.

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

Ensaio de efluxo de colesterol | Medicina | Vídeo

Cholesterol Efflux Assay

O ensaio de colesterol é concebido para quantificar a taxa de efluxo de colesterol a partir de células cultivadas e da capacidade de plasma aceitadores para aceitar o colesterol libertado a partir de células. O ensaio consiste de marcação das células com o equilíbrio do colesterol, de colesterol entre pools intracelulares e libertação de colesterol para um aceitador extracelular.

Ensaio de efluxo do 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

29.6K visualizações.  Baker IDI Heart and Diabetes Institute. O ensaio de colesterol é concebido para quantificar a taxa de efluxo de colesterol a partir de células cultivadas e da capacidade de plasma aceitadores para aceitar o colesterol libertado a partir de células. O ensaio consiste de marcação das células com o equilíbrio do colesterol, de colesterol entre pools intracelulares e libertação de colesterol para um aceitador extracelular.

Vídeo: 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.

Ensaio Metástase Experimental

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.

Em função Assay Canine Muscle Vivo

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

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.

Ensaio esferóide para TGF-β Medida induzida 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 ...

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.

Um<em&gt; In vivo</em&gt; Ensaio para ensaio de permeabilidade dos vasos sangüíneos

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.

O<em&gt; Em ovo</em&gt; CAM-ensaio como um Xenograftmodel para o 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.

Simulando pâncreas Neuroplasticidade:<em&gt; In Vitro</em&gt; Dual-neurônio Plasticidade Assay

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

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Formação Mammosphere Ensaio de cancro da mama humano tecidos e linhas celulares

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

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.

Tridimensional (3D) Tumor Esferóide 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 ...

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.

Efeitos diferenciais de drogas hipolipemiantes na modulação da morfologia das 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.