Notes techniques: <ul><li> La gélification est initiée par l&#39;ajout de sels. Par conséquent, il est essentiel que les sels ne viennent en contact avec PuraMatrix jusqu&#39;à gélification est désiré, l&#39;exposition aux sels de la force ionique très faible (par exemple, &lt;0,05 M) provoque la gélification irréversible. </li><li> Les restes de solutions de stock qui sont prêts peuvent être utilisés à une date ultérieure à condition qu&#39;ils ne l&#39;ont pas entrer en contact avec des sels. Vous n&#39;avez pas besoin de s&#39;inquiéter sonication répéter. </li><li> Les bulles d&#39;air peuvent être enlevés aliquotage PuraMatrix dans des tubes stériles suivis par centrifugation. </li><li> Soyez très prudent lors des changements de médias comme l&#39;aspiration peut facilement perturber le lit PuraMatrix. </li></ul> Matériaux: BD PuraMatrix RAD16 Peptide-1 Hydrogel% (Cat # 354250): La concentration de peptide total du mélange non dilué est de 10 mg / ml. <ul><li> Sonicator bain-marie ou Vortex </li><li> 96 puits, boîte de culture cellulaire </li><li> Milieux de culture cellulaire </li><li> Filtré stérile H 2 O </li><li> Stérile Saccharose filtrée à 20% </li><li> Cellules (tous à 5000 cellules / puits): OVCAR-5 </li></ul> Toutes les étapes doivent être effectuées dans des conditions aseptiques. Procédure: <ol><li> Soniquer l&#39;PuraMatrix 1% (RAD16) dans un sonicateur bain-marie pendant 30 minutes pour réduire la viscosité avant de l&#39;utiliser. </li><li> La solution PuraMatrix 1% peuvent désormais être répartie dans des tubes stériles pour simplifier de futures expériences. <ol class="lalpha"><li> Eviter la formation de bulles d&#39;air, si elles forment tout simplement spin bas des tubes stériles contenant PuraMatrix à 1000 rpm (300 xg) pendant 5 minutes. </li><li> La concentration de peptide total du mélange non dilué est de 10 mg / ml. </li></ol></li><li> Deux séries de quatre puits seront plaquées à 2,5 mg / ml et 1,25 mg / ml de concentration de peptide total, respectivement. </li><li> Diluer le PuraMatrix dans chaque puits mis à 0,2 um stériles sucrose filtrée à 20% de saccharose et de 13,3% au total des concentrations de peptide de 5 mg / ml et 2,5 mg / ml, respectivement pour atteindre une concentration de BD 2x PuraMatrix de 10% de saccharose. <ol class="lalpha"><li> Faire 20% de saccharose, diluer 10 g de saccharose, puis le volume à 50 ml d&#39;eau. </li><li> 13% et 10% de saccharose peut être préparé en diluant la solution stock de 20%. <ol class="lroman"><li> 13% de saccharose (pour chaque 10 ml): 6,5 ml de saccharose à 20% + 3,5 ml stériles H 2 O. </li><li> 10% de saccharose (pour chaque 10 ml): 5,0 ml de saccharose à 20% + 5,0 ml stériles H 2 O. </li></ol></li></ol></li><li> Préparer un mélange maître de peptide PuraMatrix basé sur le nombre de puits que vous l&#39;intention de la plaque. (Voir l&#39;exemple ci-dessous.) Puis aliquotes dans des tubes individuels contenant 25 ml de mélange maître. <ol class="lalpha"><li> Le master mix doit être préparé à deux fois la concentration finale souhaitée du peptide totale car il sera dilué avec un volume égal de cellules pendant le placage. <img src="/files/ftp_upload/1692/1692_fig11.jpg" alt="Figure 1" /></li></ol></li><li> Préparer les suspensions de cellules par trypsinisation cultures cellulaires monocouche cellulaire. <ol class="lalpha"><li> 2 ml de Trypsine EDTA 0,05% peut être utilisé pour chaque flacon T-75. </li></ol></li><li> Neutraliser la trypsine par addition de milieux de culture cellulaire contenant 10% inactivé par la chaleur du sérum de veau fœtal (utiliser au moins 2 ml de milieu par ml de trypsine utilisée dans l&#39;étape précédente.) </li><li> Centrifuger les cellules à 1000 rpm (~ 300 xg) pendant 5 minutes pour créer un culot cellulaire. <ol><li> Resuspendre les cellules dans 10% de saccharose filtrée stérile. </li><li> Compter les cellules et les spin-down à nouveau à 1000 rpm (~ 300 xg) pendant 5 minutes pour éliminer des traces de sel dans les médias. </li></ol></li><li> Resuspendre les cellules OVCAR-5 dans 10% de saccharose à 2,0 x 10 5 cellules / ml sorte que le nombre final de cellules par puits sera 5000 cellules dans chaque puits. Les étapes suivantes doivent être effectuées rapidement. </li><li> Ajouter 25 ul de l&#39;2,0 x 10 5 cellules / ml, suspension pour le premier tube individuels contenant 25 ul du mélange maître de PuraMatrix comme le montre le schéma ci-dessus. <ol class="lalpha"><li> Mélanger rapidement à la pipette (~ 5 fois haut et bas) dans le tube, sans introduire de bulles d&#39;air. </li><li> Puis la pipette de la cellule de 50 ul de suspension / PuraMatrix mélange dans le puits approprié d&#39;un plat de 96 puits. </li><li> Initier la gélification de la BD par PuraMatrix délicatement pipetage 150 ul milieux de culture cellulaire (RPMI + 10% de FBS) sur le côté du puits <img src="/files/ftp_upload/1692/1692_fig12.jpg" alt="Figure 2" /></li></ol></li><li> Répétez les étapes décrites dans 8 jusqu&#39;à ce que tous les puits ont été plaqués. </li><li> Après 30 minutes très doucement supprimer ~ 2 / 3 de la presse à l&#39;aide d&#39;une pipette 200 pi pour équilibrer le pH de l&#39;hydrogel. <ol class="lalpha"><li> NE PAS UTILISER un aspirateur AS Cela perturbera LA MATRICE </strong&gt; </li><li> Placez la pointe de la pipette dans le puits afin qu&#39;il ne touche pas le lit PuraMatrix et retirez délicatement les médias à partir du puits. </li><li> Des précautions doivent être prises pour ne pas perturber la matrice lors de la culture de cellules dans PuraMatrix. </li></ol></li><li> Très doucement, ajouter 150 l de frais milieux de culture cellulaire sur le côté des cellules contenant ainsi encapsulé dans PuraMatrix. </li><li> Changer le milieu de culture cellulaire dans chaque puits toutes les 48 heures avec milieu de culture frais en prenant soin de répéter les étapes 10 et 11. </li></ol> Les résultats représentatifs: <a href="https://www.jove.com/files/ftp_upload/1692/1692_fig1.jpg"><img src="/files/ftp_upload/1692/1692_fig1tmb.jpg" alt="Figure 1" border="0" /></a> Figure 1. OVCAR-5 cellules ont été encapsulé comme décrit dans le protocole ci-dessus. Les images ont été acquises en utilisant une inversé Olympus FV1000 microscope équipé de Spectra-Physics DeepSee Ti: Sapphire laser réglé à 750 nm. Séances d&#39;imagerie ont été menées sur les jours 1, 3, 5 et 7 de post-plaquage. Une longue distance de travail, de l&#39;eau plongeant objectif 40x (Olympus LUMPLFLN 0,8 NA) a été utilisé pour l&#39;image à travers le PuraMatrix. Tridimensionnelle des volumes ont été recueillies par l&#39;acquisition de piles d&#39;images dans les étapes 1,47 um axiale. Deux organisations non descanned détecteurs recueillies à l&#39;aide de l&#39;émission l&#39;autofluorescence violet (440-490 nm) et vert (510-550 nm) filtres passe-bande. Les images finales et les films pile de profondeur ont été traitées à l&#39;aide ImageJ en combinant deux canaux de détection.

<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

Plus de preuves suggèrent que les cellules cancéreuses en culture en trois dimensions avec plus de précision récapitule la complexité de la biologie de la tumeur. Beaucoup de ces modèles utilisent la membrane basale reconstituée issus d&#39;animaux qui contiennent une quantité variable de facteurs de croissance et cytokines qui peuvent influencer la croissance de ces modèles de culture cellulaire. Ici, nous décrivons en détail la préparation et l&#39;utilisation de PuraMatrix, un gel auto disponibles commercialement peptide montage qui est dépourvu de matériels d&#39;origine animale et des agents pathogènes d&#39;encapsuler et de propager la lignée cellulaire du cancer de l&#39;ovaire, OVCAR-5. Nous commençons par décrire comment préparer le PuraMatrix avant utilisation. Ensuite, nous démontrons comment bien mélanger la suspension cellulaire PuraMatrix et d&#39;encapsuler les cellules de l&#39;hydrogel. Lors de l&#39;ajout de milieux de culture cellulaire ou l&#39;injection dans un environnement physiologique, la composante du peptide PuraMatrix rapide auto assemble en un hydrogel 3D qui présente une structure fibreuse nanomètre échelle avec une taille moyenne de pores de 500 à 200 nm<sup&gt; 1</sup&gt;. En outre, nous démontrons comment propager les cultures cultivées en PuraMatrix encapsulé. Lorsque encapsulé dans PuraMatrix, OVCAR-5 cellules s&#39;assemblent en trois structures acineuses dimensions qui ressemblent plus à la morphologie des nodules micrométastatique observés dans la clinique de monocouche dans des modèles in vitro. En utilisant la microscopie confocale, nous illustrons l&#39;apparence d&#39;un représentant OVCAR-5 cellules encapsulées dans PuraMatrix le jour 1, 3, 5 et 7 plaquage poste. L&#39;utilisation de PuraMatrix aux cellules cancéreuses de la culture devrait améliorer notre compréhension de la maladie et nous permettre d&#39;évaluer la réponse au traitement dans des systèmes modèles prédictifs plus cliniquement.

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

PuraMatrix Encapsulation of Cancer Cells

Cette vidéo montre comment encapsuler et les cellules cancéreuses de la culture dans les PuraMatrix, une auto disponibles commercialement assemblage gel de peptides.

L&#39;encapsulation des cellules cancéreuses PuraMatrix

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

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16.6K vues.  Harvard Medical School. Cette vidéo montre comment encapsuler et les cellules cancéreuses de la culture dans les PuraMatrix, une auto disponibles commercialement assemblage gel de peptides.

Vidéo: PuraMatrix Encapsulation of Cancer Cells

Title Cell Encapsulation by Droplets

Titre encapsulation cellulaire par des gouttelettes

Recherche

Biologie

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.

Encapsulation Cellulaire dans les hydrogels 3D pour l&#39;ingénierie tissulaire

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

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Dérivation de cellules souches de la souris Trophoblast à partir de blastocystes

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

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.

Isolement des cellules souches de l&#39;homme xénogreffes de cancer du pancréas

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.

Isolement et culture des cellules souches épithéliales adultes de la peau humaine

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.

Purification cytométrie en flux des cellules de souris méiotique

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.

Culture ex vivo des cellules humaines primaires épithéliales des trompes de Fallope

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.

Visualisation des cellules interstitielles de Cajal (CIC) Réseau Souris

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.

Isolement des cellules immunitaires des tumeurs primitives

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.

Induction et analyse des épithéliale à mésenchymateuses transition

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