Gostaríamos de reconhecer as grandes Stem Cell FACS Centro de Pesquisa e agradecer especialmente Jessica Scholes e Felicia Codrea por sua ajuda com a classificação celular. O trabalho foi financiado pelo CIRM RN2-00904-1, K08 HL074229, da American Thoracic Society / Fundação DPOC ATS-06-065, A Fundação preocupação, a UCLA Jonsson Comprehensive Cancer Center Thoracic Oncology SPORE Cancer Program / Pulmão, da Universidade da Califórnia do Câncer Pesquisa Comissão de Coordenação e Gwynne Hazen cereja Memorial Laboratories (BG).

Esboço de Passos 1. Dissecção da traquéia 2. Limpeza traquéia e cortá-lo 3. Digestão enzimática e transformação em suspensão de célula única 4. A coloração por FACS e triagem 5. Processamento de células classificadas para in vivo e in vitro, em modelos 1. Dissecção da traquéia <ol><li> Euthanize o rato com uma injeção intraperitoneal de 0,1 cc mg/0.2 de pentobarbital. </li><li> Cortar abrir a parede abdominal, e mover os intestinos para o lado a exposição da aorta abdominal e depois dissecá-lo. </li><li> Abra o peito, cortando através do diafragma e da parede torácica anterior em ambos os lados para expor o coração e os pulmões. Continuar a cortar-se para dentro do gargalo de remover gordura e glândulas salivares para expor a traqueia. </li><li> Remover o timo e cortar o pulmão fora de suas conexões abaixo e atrás. SeparComeram a traqueia do esófago, inserindo uma pinça entre elas, em seguida, cortado a traqueia, na sua parte mais superior acima da sua ligação com a laringe. Em seguida, mantenha a traquéia com uma pinça e dissecá-lo de seu apego e continue para baixo para removê-lo com o coração e pulmão en-bloco. Cortar o coração e os pulmões próximo ao hilo para incluir a maioria dos brônquios direito e esquerdo principal. </li><li> Coloque a traqueia numa placa de Petri que continha a recolha meio em gelo. </li></ol> 2. Traquéia limpeza e corte- <ol><li> Sob o microscópio de dissecação, utilize uma pinça de ponto muito finas (n º 5) e Tübingen Vannas tesouras (FST 15003-08 finescience.com) para limpar a traquéia de todos os outros tecidos ligados a ele. Isto inclui: gânglios linfáticos, nervos laríngeo recorrente, gordura, glândula tireóide e restantes vasos pulmonares. A parte superior da traqueia requer atenção especial, para remover as cartilagens laríngeas grandes, sem comprometer o maior grupo de SMGque esteja alojada entre a cartilagem cricóide eo primeiro anel traqueal cartilaginoso (C1) 4. </li><li> Cortar a traqueia para dentro da parte superior e da parte inferior entre C4 e C5 e, em seguida, cortado ambas as partes através do lúmen da traqueia. </li></ol> 3. A digestão enzimática e Transformação em suspensão de célula única (SCS) <ol><li> Incubar a parte inferior da traqueia, em 1 ml de 16 U dispase em um tubo de Eppendorf à temperatura ambiente (RT) durante 30 minutos, como descrito anteriormente 5. Em seguida, adicionar 0,5 mg / ml de DNase I em DMEM durante mais 20 min à temperatura ambiente. </li><li> Remover as traqueias digeridas em meio fresco a recolha de uma placa de Petri esterilizada, e retirar o epitélio da superfície sob o microscópio de dissecação. Usar uma pipeta para transferir o meio contendo o epitélio evaporadas para um tubo cónico de 50 ml. </li><li> Girar o epitélio despojado a 1.000 xg, a 4 ° C. Remover o sobrenadante e adicionar 0,1% de tripsina / EDTA e incubado com agitação thtubo e em 37 ° C agitando incubadora (200 rpm) durante 30 min. Após a incubação, utilizando uma ponta de 1000 uL, pipetar para cima e para baixo para quebrar aglomerados e formar uma suspensão de células simples. </li><li> Coloque a traqueia superior, num tubo cónico contendo 1 ml de pronase 0,15%. Em seguida, incuba-se a 4 ° C durante 4 horas. </li><li> Vortex suavemente o tubo e, em seguida, colocar a traqueia para meio fresco e recolha de confirmar sob o microscópio de dissecação que haja separação completa da superfície do epitélio (SE). </li><li> Picar a traqueia com uma tesoura fina de abrir os compartimentos SMG e depois incubar durante mais uma hora à temperatura ambiente em pronase, com agitação lenta. </li><li> Girar o tecido traqueal, remover a pronase, adicionar 0,1% de tripsina / EDTA e incubado a 37 ° C durante 30 min com agitação como acima descrito. </li><li> Passagem das células digeridas, aglomerados e quaisquer partes restantes das glândulas através de agulhas 20G, 23G e 26G utilizando uma seringa de 10 ml. Repita as vezes várias passagens em cada um com o tamanho de agulha antescargo. Agora, a maioria das células deve ser em um SCS. </li><li> Filtrar a suspensão através de um filtro 40 mM em seguida, lave o coador e girar as células. Em seguida reconstituir as células no volume apropriado de meio, e contar as células imediatamente para coloração de FACS e de classificação. Não deixar as células em 4 ° C ou sobre gelo durante várias horas, pois verificou-se que isto afecta a viabilidade e reduz gravemente a esfera formando eficiência. </li></ol> 4. A coloração para FACS e classificação <ol><li> Tome células suficientes para a preparação dos tubos não coradas e único de compensação coradas então manchar o resto das células com os anticorpos primários, Trop2 cabra e de rato ITGA6, durante 15 min a RT. Em seguida, lave com PBS ou recolher médio, removedor do sobrenadante com muito cuidado e quebrar o pellet celular. </li><li> Mancha com o anticorpo secundário adequado, incubar no escuro à temperatura ambiente durante 10 min, lavar e remover o sobrenadante e em seguida romper a pelota. </li><li> Nas FACSARIA, depois de ajustar sua tensão e compensação, portão para fora dos escombros, células muito pequenas, doublets celulares e células mortas. Em seguida, a maioria das células da SE deve ser TROP2 positivo enquanto 20-50% de células SMG será TROP2 + 6. </li><li> Definir o gate de triagem para Trop2 + + ITGA6 células de células basais (SE) e para Trop2 + células (+ /-ITGA6 células positivas) de SMG (células do ducto). </li></ol> 5. Processamento células separadas para: Cultura in vitro e in vivo no modelo do 5,1 in vitro esfera formando 5-9 cultura <ol><li> Misturar 100 ul de &quot;Factor de Crescimento reduzido Matrigel&quot; com 100 ul de meio completo 10,11 (ver Tabela 1), contendo até 50.000 células seleccionadas em seguida, colocar esta mistura na câmara superior de um poço de 24 trans-poço. Em seguida, colocar 400 ul de meio completo em câmara inferior e incubar a 37 ° C. Alterar meio em câmara baixa a cada dois dias e adicionar 100 ul extra de Matrigela cada semana ou mais cedo, se torna-se fina. </li></ol> 5.2 No modelo in vivo <ol><li> Reconstituir o pellet de células classificadas em meio completo misturado 1:1 com matrigel a uma concentração de 5.000-10.000 células por 50 ul. </li><li> Anestesiar os receptores C57/BL6 ratos. Administrar carprofeno (5 mg / kg) subcutaenously como analgésico. Esterilizar e raspar as costas em seguida, fazer uma incisão na pele central longitudinal na parte superior das costas. A incisão deve ser grande o suficiente para identificar o local da injecção de gordura almofada, bem como a bolha que se forma depois da injecção. Use o lado sem corte da tesoura para alargar malotes entre a pele ea parte de trás da parede torácica em ambos os lados para maximizar o acesso e visualização. </li><li> Identificar a borda medial da escápula, empurrando o membro anterior mouse para cima e para trás para ver o movimento da escápula. Tomar 50 ul da suspensão de células para uma seringa de insulina 300 ul BD e injectá-la no corpo adiposo apenas à escápula medial e lateral, paraas vértebras. Você deve ser capaz de ver uma bolha se não você são muito profundas. Repita a mesma do outro lado, em seguida, o grampo ou o ponto de incisão na pele. Verificar ratos e dar carprofeno (5 mg / kg, SC), uma vez por dia, durante 48 hr. </li><li> Após 3 semanas, a eutanásia ratos com pentobarbatol (0,1 cc mg/0.2, IP), abra a pele como acima e dissecar a almofada toda a gordura de ambos os lados com uma margem de segurança para evitar a falta de qualquer estrutura de tecido que pode ter se formado e estendido ao redor. Fixar em PFA e embed em parafina 12. Extremo cuidado deve ser empregado durante o corte e cortar o bloco não para ignorar ou esquecer uma estrutura. A melhor prática é cortar 40 um uma vez e depois cortar uma fatia mM 4, coloque-o sobre uma lâmina de vidro e examiná-lo sob o microscópio para confirmar se existem &quot;estruturas epiteliais&quot; nesta parte do bloco ou não. Se não há nada visto, então cortar mais uma vez e repita esta etapa. </li></ol> 6. Resultados representativos Stripping das traqueias rato irá resultar numa traqueia desnudada que é visível por microscopia de luz, como mostrado na Figura 1A. Figura 1B mostra uma vista de campo claro de uma traqueia após decapagem com epitelial glândulas submucosas ser libertados a partir dos tecidos e dos cachos de uvas que se assemelham. Pronase remove o epítopo ITGA6 e é por isso que as condutas TROP2 células não podem ser separados em ITGA6 + e - as populações. No entanto, dispase, que preserva o epítopo ITGA6, digere todos epitélio da superfície e as células do ducto, mesmo com tempos de incubação muito curtos e, portanto, não pode ser utilizado para separar as células basais da conduta 3. A citometria de fluxo das suspensões de células individuais deve mostrar a frente diagramas de dispersão e de dispersão lateral, que estão representados na Figura 2A. Uma boa separação das células do ducto do resto, se as células da traqueia deve ser visto com o anticorpo Trop2 em citometria de fluxo por fluorescência de células activado sorting (FACS), como visto na Figura 2B. Esferas deve ser visível na matrigel após cerca de 1 semana em cultura e são densos em aparência, como mostrado na Figura 3. A eficiência deste processo é a ordem de 1-2%, e as células individuais ainda estará presente no matrigel e provavelmente representam células de ductos glândulas submucosas que não possuem a capacidade de auto-renovação. A injecção de células-tronco duto no rato resultados almofada de gordura na formação da glândula submucosa estruturas semelhantes que são mostrados Figura 4. Estes são vistos nos blocos, mesmo sem coloração de H &amp; E. Estes são esféricos e possuem um lúmen central. Muitas secções transversais necessitam de ser feitos por meio da almofada de gordura, a fim de não perder essas estruturas epiteliais. <img alt="Figura 1" src="/files/ftp_upload/3731/3731fig1.jpg" /> Figura 1. Digestão enzimática de epitélio traqueal. A remoção de células epiteliais da superfície é mostrada com blacsetas k, a remoção das células das glândulas submucosas dutos é mostrado com setas verdes. Traqueias desnudadas que são visíveis por microscopia de luz são apresentados após 30 min de digestão dispase e após 4 h de digestão com pronase. <img alt="A Figura 2A" src="/files/ftp_upload/3731/3731fig2A.jpg" /> Figura 2A. I. Citometria de fluxo das suspensões de células individuais que mostram dispersão frontal representante e gráficos de dispersão laterais da traquéia de rato depois de superfície epitelial stripping. ii. Representante parcelas FACS para TROP2 expressão em células do duto de traquéia de rato. <img alt="A Figura 2B" src="/files/ftp_upload/3731/3731fig2B.jpg" /> Figura 2B. I. A citometria de fluxo das suspensões de células isoladas a partir de epitélio da superfície despida de dois terços inferiores da traqueia de rato mostrando dispersão representativo para a frente e diagramas de dispersão lateral. ii. Lote típico de FACS ITGA6 expressão em surface epitélio. iii. Outra parcela FACS representante da frente e dispersão lateral do epitélio superfície mostrando volta gating da população de células basais em verde. iv. ITGA6 típico e TROP2 expressar população de células em verde. <img alt="Figura 4" src="/files/ftp_upload/3731/3731fig3.jpg" /> Figura 3. Evolução temporal do desenvolvimento das esferas. A. esferas de condutas. Esferas deve ser visível na matrigel após cerca de 1 semana de cultura e quase todos são densos em aparência. As células individuais que não formam esferas são também observadas. (Barra de escala = 50 mm). Esferas B. basais são maiores e luminal na aparência. As células individuais que não formam esferas são também observadas. (Barra de escala = 50 mm). <img alt="Figura 4" src="/files/ftp_upload/3731/3731fig4.jpg" /> Figura 4. A injecção de células-tronco duto no rato resultados almofada de gordura na formação da glândula submucosa estruturas semelhantes. Estruturas representativassão mostrados na almofada de gordura e alguns são apresentados produzindo mucina (azul claro), com o ácido de Schiff periódico mancha azul Alcian.

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Esta técnica para isolar as células do ducto e basal das vias aéreas é importante para melhorar a compreensão de reparação e regeneração das vias aéreas e de doenças das vias respiratórias. As técnicas descritas aqui incluem alguns passos críticos. O primeiro é o período de digestão enzimática optimizado. A segunda é a criação de uma suspensão de células individuais através de passagens em série com agulhas calibre progressivamente mais elevadas para impedir ruptura da célula, mas a romper-se g...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Conclua médio 10: DMEM-F-12, 50/50, 1X) </td><td> Mediatech </td><td> 15-090 CV- </td></tr><tr><td> Hepes (15 mM) </td><td> Invitrogen </td><td> 15630 </td></tr><tr><td> Bicarbonato de sódio (3,6 mM ou 0,03%) </td><td> Invitrogen </td><td> 25080 </td></tr><tr><td> L-glutamina (4 mM) </td><td> Mediatech </td><td> 25-005 Cl- </td></tr><tr><td> Penicilina (100 U / ml) </td><td> Mediatech </td><td> 30-001 CI- </td></tr><tr><td> Estreptomicina (100 ug / m) </td><td> Mediatech </td><td> 30-001 CI- </td></tr><tr><td> Anfotericina B (0,25 ug / ml) </td><td> Lonza </td><td> 17-836R </td></tr><tr><td> EmSulin (10 ug / ml) </td><td> Sigma </td><td> I6634 </td></tr><tr><td> Transferrina (5 ug / ml) </td><td> Sigma </td><td> T1147 </td></tr><tr><td> Toxina da cólera (0,1 ng / ml) </td><td> Sigma </td><td> C8052 </td></tr><tr><td> Factor de Crescimento Epidérmico (25 ng / ml) </td><td> BD </td><td> 354001 </td></tr><tr><td> Extrato de Pituitária Bovina (30 ug / ml) </td><td> Invitrogen </td><td> 13028-014 </td></tr><tr><td> O soro de bovino fetal (5%) </td><td> Pescador </td><td> SH3008803HI </td></tr><tr><td> Ácido retinóico (0,05 uM) </td><td> Sigma </td><td> R2625 </td></tr><tr><td> Fator de Crescimento reduzido Matrigel </td><td> BD </td><td> 354230 </td></tr></tbody></table> Tabela 1. Conclua componentes de mídia. <table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td&gt; <td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Pronase </td><td> Roche </td><td> 10165921001 </td><td> Usado em 0,15%: -O / n a 4 ° C para isolar a digestão total de células para a cultura de traqueia (ALI) Digestão hr -4 4 ° C para isolar SMG </td></tr><tr><td> Dispase </td><td> BD Biosciences </td><td> 354235 </td><td> Usado menos 16 Unidades: 30 min à TA </td></tr><tr><td> DNase I </td><td> Sigma </td><td> DN25 </td><td> Utilizada a 0,5 mg / ml: 20-30 min à temperatura ambiente </td></tr></tbody></table> Enzimas Tabela 2. Usado para a digestão enzimática da traqueia.

isolation of basal cells and submucosal gland duct cells from mouse trachea

Grandes vias aéreas estão diretamente em contato com o meio ambiente e, portanto, suscetível a lesões de toxinas e agentes infecciosos que respiramos em 1. Grandes vias aéreas, portanto, exigem um mecanismo de reparo eficiente para proteger os nossos corpos. Este processo de reparo ocorre a partir de células-tronco nas vias aéreas e isolar essas células-tronco das vias aéreas é importante para compreender os mecanismos de reparo e regeneração. Também é importante para a compreensão de reparação anormal que pode conduzir a doenças das vias aéreas 2. O objectivo deste método é para isolar uma população de células estaminais a partir de novo os ratos ductos de glândulas submucosas traqueal e para colocar estas células in vitro e em sistemas de modelos in vivo para identificar os mecanismos de reparação e regeneração das glândulas submucosas 3. Isto mostra a produção de métodos que podem ser utilizados para isolar e ensaio das células estaminais e do ducto basal das vias aéreas grandes 3. Isto permitirápara o estudo de doenças das vias respiratórias, como a fibrose cística, asma e doença pulmonar obstrutiva crónica. Actualmente, não existem métodos para o isolamento de células das glândulas submucosas duto e não existem modelos in vivo para estudar a regeneração de glândulas submucosas.

Watch this Scientific Journal Video about Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea at JoVE.com

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

Aqui demonstramos nosso protocolo para isolamento de células basais e das glândulas submucosas duto de traqueias de rato. Nós também demonstram o método de injecção de células estaminais na almofada de gordura dorsal do rato para criar um<em&gt; In vivo</em&gt; Modelo de regeneração da glândula submucosa.

Isolamento de células basais e células Duct submucosa Glândulas de Rato Traquéia

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. ...

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JoVE Journal

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13.3K visualizações.  David Geffen School of Medicine at UCLA. Aqui demonstramos nosso protocolo para isolamento de células basais e das glândulas submucosas duto de traqueias de rato. Nós também demonstram o método de injecção de células estaminais na almofada de gordura dorsal do rato para criar um In vivo Modelo de regeneração da glândula submucosa.

Vídeo: Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolamento de células T CD4 + a partir dos nós linfáticos rato Usando Miltenyi Purificação MACS

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Biologia

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Isolamento e derivação de células embrionárias de rato Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

Isolamento de Células-Tronco da retina do olho do rato

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Isolamento de Células-Tronco do rato das Glândulas Salivares

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Isolamento e cultura de células da zona nefrogênica do rim do rato embrionárias

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Isolamento de células imunes de tumores primários

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Geração de camundongos derivadas de células-tronco pluripotentes induzidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Indução e Análise de epitelial para mesenquimal Transição

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolamento e Cultura da Dental epiteliais células-tronco do Adulto Rato Incisivo

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Isolamento e Análise Funcional de mitocôndrias de células cultivadas e mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...