Notas técnicas: <ul><li> Gelificação é iniciada pela adição de sais. Portanto, é fundamental que não sais de entrar em contato com PuraMatrix até gelificação é desejado; exposição a sais de muito baixa força iônica (eg, &lt;0,05 M) fará com que gelificação irreversível. </li><li> Soluções estoque restante que está preparado pode ser usado em uma data posterior, desde que não tenham entrado em contato com sais. Você não precisa se preocupar com sonicação repetir. </li><li> Bolhas de ar podem ser removidas PuraMatrix alíquotas em tubos estéreis, seguido por centrifugação. </li><li> Tenha muito cuidado durante as mudanças de mídia como aspiração podem facilmente perturbar a cama PuraMatrix. </li></ul> Materiais: BD PuraMatrix Peptide Hydrogel RAD16-1% (Cat # 354250): A concentração de peptídeo total da mistura não diluída é de 10 mg / ml. <ul><li> Sonicador água Banheira ou Vortex </li><li> De 96 poços prato de cultura de células </li><li> Meios de Cultura de células </li><li> Estéril Filtrado H 2 O </li><li> Sacarose filtrado estéril 20% </li><li> Células (todos menos 5.000 células / poço): OVCAR-5 </li></ul> Todos os passos devem ser realizados sob condições assépticas. Procedimento: <ol><li> Sonicate o PuraMatrix 1% (RAD16) em um sonicador banho-maria durante 30 minutos para reduzir a viscosidade antes do uso. </li><li> A solução PuraMatrix 1% pode agora ser aliquotado em tubos estéreis a simplificar experiências futuras. <ol class="lalpha"><li> Evitar a formação de bolhas de ar, se elas formam simplesmente girar os tubos estéreis, contendo PuraMatrix a 1000 rpm (300 xg) por 5 minutos. </li><li> A concentração de peptídeo total da mistura não diluída é de 10 mg / ml. </li></ol></li><li> Dois conjuntos de quatro poços serão banhados em 2,5 mg / ml e 1,25 mg / ml de concentração de peptídeo total, respectivamente. </li><li> Diluir o PuraMatrix em cada bem definido com 0,2 m estéril filtrado sacarose 20% e sacarose 13,3% para concentrações totais de peptídeo de 5 mg / ml e 2,5 mg / ml, respectivamente, para alcançar uma concentração 2x de BD PuraMatrix em sacarose 10%. <ol class="lalpha"><li> Fazer sacarose 20%, diluir 10 g de sacarose, em seguida, o volume a 50 ml de água. </li><li> 13% de sacarose e 10% podem ser preparadas por diluição da solução estoque de 20%. <ol class="lroman"><li> 13% de sacarose (para cada 10 ml): 6,5 ml de sacarose 20% + 3,5 ml estéril H 2 O. </li><li> 10% Sacarose (para cada 10 ml): 5,0 ml de sacarose 20% + 5,0 ml estéril H 2 O. </li></ol></li></ol></li><li> Prepare uma mistura mestre de peptídeo PuraMatrix com base no número de poços que pretende prato. (Veja o exemplo abaixo.) Então alíquotas em tubos individuais contendo 25 ml da mistura de mestre. <ol class="lalpha"><li> O master mix deve ser preparado com o dobro da concentração final desejada de peptídeo total como ele vai ser diluído com igual volume de células durante a galvanização. <img src="/files/ftp_upload/1692/1692_fig11.jpg" alt="figura 1" /></li></ol></li><li> Preparar suspensões de células por trypsinizing culturas de células de células em monocamada. <ol class="lalpha"><li> 2 ml de 0,05% Tripsina EDTA pode ser usado para cada Flask T-75. </li></ol></li><li> Neutralizar a tripsina com a adição de meios de cultura celular contendo 10% de calor soro fetal bovino inativado (uso pelo menos 2 ml de mídia por ml de tripsina usada na etapa anterior.) </li><li> Centrifugar as células a 1000 rpm (~ 300 xg) por 5 minutos para criar um pellet de células. <ol><li> Ressuspender as células em 10% de sacarose estéril filtrado. </li><li> Contar as células e spin para baixo novamente a 1000 rpm (~ 300 xg) por 5 minutos para remover traços de sal encontrados na mídia. </li></ol></li><li> Ressuspender as células OVCAR-5 em sacarose 10% a 2.0 x 10 5 células / ml para que a contagem de células por poço finais serão 5.000 células em cada poço. Os seguintes passos devem ser realizados rapidamente. </li><li> Adicionar 25 mL do 2,0 x 10 5 células / ml de suspensão para o primeiro tubo contendo 25 mL individuais do mix de mestre de PuraMatrix como mostrado no esquema acima. <ol class="lalpha"><li> Rapidamente mix por pipetagem (~ 5 vezes para cima e para baixo) no tubo sem a introdução de bolhas de ar. </li><li> Em seguida, pipetar 50 ul da célula mistura suspensão / PuraMatrix no poço apropriado de um prato de 96 poços. </li><li> Iniciar a gelificação do PuraMatrix BD com cuidado pipetando 150 media de células mL de cultura (RPMI + 10% FBS) para o lado do bem <img src="/files/ftp_upload/1692/1692_fig12.jpg" alt="figura 2" /></li></ol></li><li> Repita os passos descritos no 8 até que todos os poços foram banhados. </li><li> Depois de 30 minutos Muito Remova cuidadosamente ~ 2 / 3 dos meios de comunicação utilizando uma pipeta 200 mL para equilibrar o pH do hidrogel. <ol class="lalpha"><li> NÃO USE A aspirador a vácuo como isso irá interromper o MATRIX </li><li> Coloque a ponta da pipeta no poço, para queele não toque na cama PuraMatrix e delicadamente remova a mídia do poço. </li><li> Cuidados devem ser tomados para não perturbar a matriz durante a cultura de células em PuraMatrix. </li></ol></li><li> Muito delicadamente adicionar 150 mL de meio de cultura de células frescas ao lado das células que contêm bem encapsulado em PuraMatrix. </li><li> Alterar a mídia de cultura de células em cada poço a cada 48 horas, com meios de cultura fresco com cuidado repetindo os passos 10 e 11. </li></ol> Resultados representativos: <img src="/files/ftp_upload/1692/1692_fig1tmb.jpg" alt="figura 1" border="0" /> Figura 1. OVCAR-5 células foram encapsulados como descrito no protocolo acima. Imagens foram adquiridas usando uma invertida Olympus FV1000 microscópio equipado com Ti-DeepSee Spectra Physics: Sapphire a laser ajustado para 750 nm. Sessões de imagem foram realizados nos dias 1, 3, 5 e 7 pós-plating. A longa distância de trabalho de água, mergulhando objetiva de 40x (Olympus LUMPLFLN 0,8 NA) foi usado para a imagem através da PuraMatrix. Volumes tridimensionais foram coletados através da aquisição de pilhas de imagens em 1,47 mM passos axial. Duas organizações não-descanned detectores coletados a emissão autofluorescência utilizando violeta (440-490 nm) e verde (510-550 nm) filtros passa-banda. As imagens finais e filmes pilha de profundidade foram processadas utilizando ImageJ, combinando ambos os canais de detecção. Por favor, <a href="https://www.jove.com/files/ftp_upload/1692/1692_fig1.jpg">clique aqui</a> para ver uma versão ampliada da figura 1.

<ol>
	<li>Gelain, F., Bottai, D., Vescovi, A., Zhang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designer+self-assembling+Peptide+nanofiber+scaffolds+for+adult+mouse+neural+stem+cell+3-dimensional+cultures.">Designer self-assembling Peptide nanofiber scaffolds for adult mouse neural stem cell 3-dimensional cultures.</a> PLoS One. 1, e119-e119 (2006).</li></ol>

puramatrix encapsulation of cancer cells

Crescente evidência sugere que as células cancerosas cultura em três dimensões com mais precisão recapitula a complexidade da biologia tumoral. Muitos destes modelos utilizam membrana basal reconstituída derivados de animais que contêm uma quantidade variável de fatores de crescimento e citocinas que podem influenciar o crescimento desses modelos de cultura de células. Aqui, descrevemos em detalhes a preparação eo uso de PuraMatrix, um disponível comercialmente auto gel montagem peptídeo que é desprovido de materiais de origem animal e patógenos para encapsular e propagar a linha de células de câncer de ovário, OVCAR-5. Começamos por descrever como preparar o PuraMatrix antes do uso. Em seguida, demonstrar como misturar corretamente o PuraMatrix e suspensão de células para encapsular as células do hidrogel. Após a adição de meios de cultura celular ou injeção em um ambiente fisiológico, o componente de peptídeo PuraMatrix rapidamente auto monta em um hidrogel 3D que exibe um nanômetro estrutura fibrosa escala com um tamanho médio dos poros de 500-200 nm<sup&gt; 1</sup&gt;. Além disso, demonstramos como propagar culturas cultivadas em PuraMatrix encapsulados. Quando encapsulado em PuraMatrix, OVCAR-5 células reúnem-se em três estruturas acinares dimensional que mais se assemelham a morfologia dos nódulos micrometastático observados na prática clínica de monocamada em modelos in vitro. Usando a microscopia confocal, ilustramos o surgimento de representante OVCAR-5 células encapsuladas em PuraMatrix no dia 1, 3, 5 e 7 plating post. O uso de PuraMatrix às células cancerosas cultura deve melhorar a nossa compreensão da doença e permitem avaliar a resposta ao tratamento em sistemas modelo mais preditivo clinicamente.

Watch this Scientific Journal Video about PuraMatrix Encapsulation of Cancer Cells at JoVE.com

PuraMatrix Encapsulation of Cancer Cells

Este vídeo demonstra como para encapsular e células cancerosas em cultura PuraMatrix, um auto comercialmente disponíveis montagem gel peptídeo.

Encapsulation PuraMatrix de células de câncer

Increasing evidence suggests that culturing cancer cells in three dimensions more accurately recapitulates the complexity of tumor biology. Many of these ...

Universidad San Sebastian

Research

JoVE Journal

Biology

16.6K visualizações.  Harvard Medical School. Este vídeo demonstra como para encapsular e células cancerosas em cultura PuraMatrix, um auto comercialmente disponíveis montagem gel peptídeo.

Vídeo: PuraMatrix Encapsulation of Cancer Cells

Title Cell Encapsulation by Droplets

Encapsulation título celular por Droplets

Biologia

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Isolamento e Análise de Células-Tronco Hematopoéticas da Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

O encapsulamento celular em Hidrogéis 3D para Engenharia de Tecidos

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

Isolamento de células-tronco de xenoenxertos Humano do Câncer de pâncreas

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolamento e Cultura de Células-Tronco Adultas epitelial da pele humana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Purificação de Citometria de Fluxo de células de camundongo meiótica

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Ex Vivo de Cultura Primária células epiteliais trompa de Falópio

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

Visualização das células intersticiais de Cajal Network (ICC) em ratos

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

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 ...

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 ...