Note tecniche: <ul><li> Gelificazione è iniziata con l&#39;aggiunta di sali. Pertanto, è fondamentale che i sali non vengono a contatto con PuraMatrix fino a gelificazione è desiderato, l&#39;esposizione a sali di molto bassa forza ionica (ad esempio, &lt;0,05 M) causerà gelificazione irreversibile. </li><li> Soluzioni rimanenze che vengono preparati possono essere usati in un secondo momento a condizione che non sono venuto in contatto con i sali. Non è necessario preoccuparsi di sonicazione ripetere. </li><li> Le bolle d&#39;aria può essere rimosso aliquoting PuraMatrix in provette sterili seguita da centrifugazione. </li><li> Prestate molta attenzione durante i cambi di mezzi di comunicazione come l&#39;aspirazione può facilmente interrompere il letto PuraMatrix. </li></ul> Materiali: BD PuraMatrix Peptide RAD16 Hydrogel-1% (Cat # 354250): la concentrazione del peptide totale della miscela non diluito è di 10 mg / ml. <ul><li> Acqua Sonicator Vasca o Vortex </li><li> 96-bene delle cellule piatto cultura </li><li> Colture Cellulari media </li><li> Sterile filtrata H 2 O </li><li> Saccarosio sterile filtrata 20% </li><li> Cellule (il tutto a 5.000 cellule / pozzetto): OVCAR-5 </li></ul> Tutte le fasi devono essere eseguite in condizioni asettiche. Procedimento: <ol><li> Sonicare PuraMatrix l&#39;1% (RAD16) in un sonicatore acqua del bagno per 30 minuti per ridurre la viscosità prima dell&#39;uso. </li><li> La soluzione PuraMatrix 1% può essere aliquotati in provette sterili per semplificare futuri esperimenti. <ol class="lalpha"><li> Evitare la formazione di bolle d&#39;aria, se si tratta di semplicemente rallenta il provette sterili contenenti PuraMatrix a 1000 rpm (300 xg) per 5 minuti. </li><li> La concentrazione del peptide totale della miscela non diluito è di 10 mg / ml. </li></ol></li><li> Due set di 4 pozzi saranno placcato a 2,5 mg / ml e 1,25 mg / ml di concentrazione del peptide totale, rispettivamente. </li><li> Diluire il PuraMatrix in ciascun pozzetto insieme con 0,2 micron sterile filtrata saccarosio 20% e il 13,3% di saccarosio al totale delle concentrazioni del peptide di 5 mg / ml e 2,5 mg / ml, rispettivamente, per ottenere una concentrazione di 2x BD PuraMatrix nel 10% di saccarosio. <ol class="lalpha"><li> Fare il 20% di saccarosio, diluire 10 g di saccarosio, poi volume di 50 ml di acqua. </li><li> 13% e il 10% di saccarosio può essere preparato diluendo la soluzione madre al 20%. <ol class="lroman"><li> 13% di saccarosio (per ogni ml 10): 6,5 ml di saccarosio al 20% + 3,5 ml sterili H 2 O. </li><li> 10% di saccarosio (per ogni ml 10): 5,0 ml di saccarosio al 20% + 5.0 ml sterile H 2 O. </li></ol></li></ol></li><li> Preparare una master mix di peptidi PuraMatrix in base al numero di pozzi che si intende piatto. (Vedi esempio sotto). Poi aliquota in singoli tubi contenenti 25 ml della master mix. <ol class="lalpha"><li> Il master mix dovrebbe essere preparato a due volte la concentrazione finale desiderata di peptide totale in quanto sarà diluito con un uguale volume di cellule durante la placcatura. <img src="/files/ftp_upload/1692/1692_fig11.jpg" alt="figura 1" /></li></ol></li><li> Preparare sospensioni cellulari da trypsinizing cellula colture cellulari monostrato. <ol class="lalpha"><li> 2 ml di 0,05% tripsina EDTA può essere utilizzato per ogni T-75 Flask. </li></ol></li><li> Neutralizzare la tripsina con l&#39;aggiunta dei terreni di coltura cellulare contenente il 10% inattivato con il calore di siero fetale bovino (uso almeno 2 ml di mezzi per ml di tripsina utilizzato nel passaggio precedente). </li><li> Centrifugare le cellule a 1000 giri (~ 300 xg) per 5 minuti per creare un pellet di cellule. <ol><li> Risospendere le cellule nel 10% di saccarosio sterile filtrata. </li><li> Contare le celle e girare di nuovo a 1000 giri (~ 300 xg) per 5 minuti per rimuovere tracce di sale trovato nei media. </li></ol></li><li> Risospendere il OVCAR-5 cellule in 10% di saccarosio a 2,0 x 10 5 cellule / ml, in modo che il conteggio finale delle cellule per pozzetto saranno 5.000 cellule in ciascun pozzetto. Le seguenti operazioni devono essere eseguite rapidamente. </li><li> Aggiungere 25 microlitri di 2.0 x 10 5 cellule / ml di sospensione per il primo tubo singolo contenente 25 microlitri della master mix di PuraMatrix come indicato nello schema di cui sopra. <ol class="lalpha"><li> Mescolare rapidamente pipettando (~ 5 volte su e giù) nel tubo senza introdurre bolle d&#39;aria. </li><li> Pipettare quindi i 50 microlitri di cellule sospensione / PuraMatrix miscela nel pozzo appropriato di un piatto ben 96. </li><li> Avviare gelificazione del PuraMatrix BD pipettando gentilmente 150 microlitri terreni di coltura cellulare (RPMI + 10% FBS) al lato del bene <img src="/files/ftp_upload/1692/1692_fig12.jpg" alt="figura 2" /></li></ol></li><li> Ripetere la procedura descritta in 8 fino a quando tutti i pozzetti sono stati placcati. </li><li> Dopo 30 minuti molto delicatamente rimuovere ~ 2 / 3 della media con una pipetta 200 microlitri per equilibrare il pH del idrogel. <ol class="lalpha"><li> NON UTILIZZARE UN VUOTO IN QUESTO ASPIRATORE ostacolerà la MATRIX </li><li> Posizionare la punta della pipetta nel pozzetto in modo chenon toccare il letto PuraMatrix e delicatamente rimuovere il supporto dal pozzo. </li><li> Si deve prestare attenzione a non disturbare la matrice durante la coltura di cellule in PuraMatrix. </li></ol></li><li> Molto delicatamente aggiungere 150 ml di mezzi freschi colture cellulari al lato del pozzetto contenente cellule incapsulate in PuraMatrix. </li><li> Cambiare il terreni di coltura di cellule in ciascun pozzetto ogni 48 ore con terreni di coltura fresco con attenzione ripetendo i punti 10 e 11. </li></ol> Rappresentante dei risultati: <img src="/files/ftp_upload/1692/1692_fig1tmb.jpg" alt="figura 1" border="0" /> Figura 1. OVCAR-5 cellule sono state incapsulate, come descritto nel protocollo di cui sopra. Immagini sono state acquisite utilizzando un microscopio rovesciato Olympus FV1000 dotato di Spectra-Physics DeepSee Ti: zaffiro laser sintonizzati a 750 nm. Sessioni di imaging sono state condotte nei giorni 1, 3, 5 e 7 post-placcatura. A distanza di lavoro, l&#39;acqua immergendo obiettivo 40x (Olympus LUMPLFLN 0,8 NA) è stato utilizzato per l&#39;immagine attraverso la PuraMatrix. Volumi tridimensionali sono stati raccolti attraverso l&#39;acquisizione di immagini in stack 1,47 micron passi assiale. Due non-descanned rivelatori di raccolti tramite l&#39;emissione di autofluorescenza violetto (440-490 nm) e verde (510-550 nm) Filtri a banda passante. Le immagini finali e filmati stack di profondità sono stati elaborati utilizzando ImageJ unendo entrambi i canali di rilevazione. Si prega di <a href="https://www.jove.com/files/ftp_upload/1692/1692_fig1.jpg">cliccare qui</a> per vedere una versione più grande della 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 evidenza suggerisce che le cellule tumorali coltura in tre dimensioni con maggiore precisione ricapitola la complessità della biologia del tumore. Molti di questi modelli utilizzano membrane ricostituito scantinato derivati ​​da animali che contengono una quantità variabile di fattori di crescita e di citochine che possono influenzare la crescita dei modelli di cultura queste cellule. Qui, descriviamo in dettaglio la preparazione e l&#39;uso di PuraMatrix, disponibile in commercio gel peptide auto assemblaggio che è privo di materiale di origine animale e agenti patogeni a integrare e propagare il cancro ovarico linea cellulare, OVCAR-5. Iniziamo descrivendo come preparare il PuraMatrix prima dell&#39;uso. Successivamente, dimostriamo come mixare correttamente il PuraMatrix e sospensione cellulare per incapsulare le cellule in idrogel. Al momento l&#39;aggiunta di terreni di coltura cellulare o iniezione in un ambiente fisiologico, il componente del peptide PuraMatrix rapidamente auto assembla in un idrogel 3D che mostra una scala nanometrica struttura fibrosa con una dimensione dei pori media di 500-200 nm<sup&gt; 1</sup&gt;. Inoltre, ci mostrano come si propagano le culture cresciute in PuraMatrix incapsulato. Quando incapsulato in PuraMatrix, OVCAR-5 cellule assemblano in tre strutture acinose dimensionale che si avvicinano maggiormente la morfologia dei noduli micrometastatica osservati nella clinica di monostrato in modelli in vitro. Usando la microscopia confocale ci illustrano l&#39;aspetto di rappresentante OVCAR-5 cellule incapsulate in PuraMatrix il giorno 1, 3, 5, e 7 placcatura post. L&#39;uso di PuraMatrix alle cellule tumorali cultura dovrebbe migliorare la nostra comprensione della malattia e ci permettono di valutare la risposta al trattamento in sistemi modello più clinicamente predittiva.

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

PuraMatrix Encapsulation of Cancer Cells

Questo video dimostra come incapsulare le cellule tumorali e cultura in PuraMatrix, un assemblaggio di auto in commercio gel peptide.

Incapsulamento PuraMatrix delle cellule tumorali

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

Research

JoVE Journal

Biology

16.6K Views.  Harvard Medical School. Questo video dimostra come incapsulare le cellule tumorali e cultura in PuraMatrix, un assemblaggio di auto in commercio gel peptide.

Video: PuraMatrix Encapsulation of Cancer Cells

Title Cell Encapsulation by Droplets

Titolo Incapsulamento cella di goccioline

Ricerca

Biologia

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.

Incapsulamento cellulare in idrogel 3D per l&#39;ingegneria tissutale

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.

Derivazione di cellule staminali trofoblasto mouse da blastocisti

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.

Isolamento di cellule staminali da xenotrapianti di cancro pancreatico umano

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 la coltura di cellule staminali epiteliali da pelle umana

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.

Purificazione Citometria a flusso di cellule di topo Meiotic

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 Cultura Vivo di cellule primarie umane tube di Falloppio epiteliale

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.

Visualizzazione delle cellule interstiziali di Cajal (ICC) di rete in topi

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 di cellule immunitarie di tumori primari

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.

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

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