Technische Hinweise: <ul><li> Die Gelierung wird durch die Zugabe von Salzen initiiert. Daher ist es entscheidend, dass keine Salze in Kontakt mit PuraMatrix kommen, bis Gelierung gewünscht wird; Exposition gegenüber Salzen sehr niedriger Ionenstärke (zB &lt;0,05 M) wird irreversiblen Gelierung führen. </li><li> Restbestände Lösungen, die bereit sind, können zu einem späteren Zeitpunkt, sofern sie nicht in Kontakt mit Salzen kommen, verwendet werden. Sie brauchen nicht zu wiederholen, Beschallung sorgen. </li><li> Luftblasen entfernt Aliquotierung PuraMatrix in sterilen Röhrchen durch Zentrifugation gefolgt werden. </li><li> Seien Sie sehr vorsichtig bei der Medien ändert sich wie Aspiration leicht stören können die PuraMatrix Bett. </li></ul> Materialien: BD PuraMatrix RAD16 Peptide Hydrogel-1% (Kat. Nr. 354250): Die gesamte Peptid-Konzentration der unverdünnten Mischung 10 mg / ml. <ul><li> Wasser Ultraschallbad oder Vortex </li><li> 96-well Zellkulturschale </li><li> Zellkulturmedien </li><li> Sterilfiltriert H 2 O </li><li> Steriles gefiltertes 20% Saccharose </li><li> Cells (alle bei 5.000 Zellen / Well): OVCAR-5 </li></ul> Alle Schritte müssen unter aseptischen Bedingungen durchgeführt werden. Vorgehensweise: <ol><li> Mit Ultraschall die 1%-PuraMatrix (RAD16) in einem Wasserbad Sonicator für 30 Minuten, um die Viskosität vor zu reduzieren, um zu verwenden. </li><li> Die 1% PuraMatrix Lösung kann nun in sterilen Röhrchen aliquotiert werden, um zukünftige Experimente zu vereinfachen. <ol class="lalpha"><li> Vermeiden Sie die Bildung von Luftblasen, wenn sie bilden einfach Spin-Down der sterilen Röhrchen PuraMatrix bei 1000 rpm (300 xg) für 5 Minuten. </li><li> Die gesamte Peptid-Konzentration der unverdünnten Mischung 10 mg / ml. </li></ol></li><li> Zwei Sätze von 4 Bohrungen werden auf 2,5 mg / ml und 1,25 mg / ml Gesamt-Peptid-Konzentration bzw. beschichtet werden. </li><li> Verdünnen Sie die PuraMatrix in jede Vertiefung mit 0,2 mu m sterilfiltriert 20% Saccharose und 13,3% Saccharose zu insgesamt Peptid-Konzentrationen von 5 mg / ml und 2,5 mg / ml eingestellt, jeweils mit einer Konzentration von 2x BD PuraMatrix in 10% Saccharose zu erreichen. <ol class="lalpha"><li> To Make 20% Saccharose, verdünnte 10 g Saccharose, dann ein Volumen von 50 ml Wasser. </li><li> 13% und 10% Saccharose kann durch Verdünnen der 20% Stammlösung hergestellt werden. <ol class="lroman"><li> 13% Saccharose (je 10 ml): 6,5 ml 20% Saccharose + 3,5 ml sterilem H 2 O. </li><li> 10% Saccharose (je 10 ml): 5,0 ml 20% Saccharose + 5,0 ml sterilem H 2 O. </li></ol></li></ol></li><li> Bereiten Sie einen Master-Mix von PuraMatrix Peptids auf die Anzahl der Bohrlöcher Sie beabsichtigen, Platte basiert. (Siehe Beispiel unten.) Dann Aliquot in einzelne Röhrchen mit 25 ml des Master-Mix. <ol class="lalpha"><li> Der Master-Mix sollte auf das Doppelte der gewünschten Endkonzentration von insgesamt Peptid hergestellt werden, wie es mit dem gleichen Volumen von Zellen während der Beschichtung verdünnt werden. <img src="/files/ftp_upload/1692/1692_fig11.jpg" alt="Abbildung 1" /></li></ol></li><li> Bereiten Zellsuspensionen von trypsinizing Zellmonolayer Zellkulturen. <ol class="lalpha"><li> 2 ml 0,05% Trypsin EDTA kann für jede T-75 Fläschchen verwendet werden. </li></ol></li><li> Neutralisieren des Trypsins durch Zugabe von Zellkulturmedium mit 10% Hitze-inaktiviertem fetalem Rinderserum (mind. 2 ml Medium pro ml Trypsin in den vorherigen Schritt.) </li><li> Zentrifugieren Sie die Zellen bei 1000 rpm (~ 300 xg) für 5 Minuten, um sich ein Zellpellet zu schaffen. <ol><li> Resuspendieren der Zellen in 10% sterilfiltriert Saccharose. </li><li> Zählen Sie die Zellen und Spin-down wieder bei 1000 Umdrehungen pro Minute (~ 300 xg) für 5 Minuten, um Spuren von Salz in den Medien gefunden zu entfernen. </li></ol></li><li> Resuspendieren OVCAR-5-Zellen in 10% Saccharose bei 2,0 x 10 5 Zellen / ml, so dass die endgültige Zellzahl pro Well werden 5.000 Zellen werden in jede Vertiefung. Die folgenden Schritte sollten zügig durchgeführt werden. </li><li> Add 25 ul des 2,0 x 10 5 Zellen / ml Suspension zum ersten Individuum Röhrchen mit 25 ul des Master-Mix aus PuraMatrix wie in den oben schematisch dargestellt. <ol class="lalpha"><li> Schnell durch Pipettieren (~ 5 mal rauf und runter) Mischung in die Röhre, ohne dabei Luftblasen. </li><li> Dann Pipette die 50 ul Zellsuspension / PuraMatrix Mischung in die entsprechende Vertiefung einer 96-Well-Schale. </li><li> Initiieren Gelierung der BD PuraMatrix durch vorsichtiges Pipettieren 150 ul Zellkulturmedien (RPMI + 10% FBS) an der Seite des Brunnens <img src="/files/ftp_upload/1692/1692_fig12.jpg" alt="Abbildung 2" /></li></ol></li><li> Wiederholen Sie die Schritte in 8 dargestellt, bis alle Vertiefungen haben vergoldet worden. </li><li> Nach 30 Minuten ganz sanft zu entfernen ~ 2 / 3 der Medien mit einer 200 ul Pipette auf den pH-Wert des Hydrogels Gleichgewicht zu bringen. <ol class="lalpha"><li> DO NOT USE A VACUUM Aspirator AS THIS THE MATRIX gestört werden </li><li> Setzen Sie die Pipettenspitze in den Brunnen, so dasses berührt nicht die PuraMatrix Bett und entfernen Sie vorsichtig Medien aus dem Brunnen. </li><li> Es sollte darauf geachtet werden, nicht stören die Matrix während der Kultivierung von Zellen in PuraMatrix werden. </li></ol></li><li> Sehr vorsichtig Fügen Sie 150 ml frisches Zellkulturmedium auf der Seite der auch mit mehreren Zellen in PuraMatrix gekapselt. </li><li> Ändern Sie den Zellkulturmedien in jede Vertiefung alle 48 Stunden mit frischem Kulturmedium durch vorsichtiges Wiederholung der Schritte 10 und 11. </li></ol> Repräsentative Ergebnisse: <img src="/files/ftp_upload/1692/1692_fig1tmb.jpg" alt="Abbildung 1" border="0" /> Abbildung 1. OVCAR-5-Zellen eingekapselt wie in dem oben beschriebenen Protokoll. Die Bilder wurden mit einem inversen Olympus FV1000 Mikroskop mit Spectra-Physics DeepSee Ti ausgestattet: Saphir-Lasers abgestimmt, um 750 nm. Imaging-Sitzungen wurden an den Tagen 1, 3, 5 und 7 post-Beschichtung durchgeführt. Ein langer Arbeitsabstand, Wasser getaucht 40x (Olympus LUMPLFLN 0,8 NA) wurde zu Bild durch die PuraMatrix verwendet. Dreidimensionale Volumina wurden durch den Erwerb von Bildstapel in 1,47 um axiale Schritte gesammelt. Zwei nicht-descanned Detektoren gesammelt die Autofluoreszenz-Emission mit Violett (440-490 nm) und Grün (510-550 nm) Bandpass-Filter. Die endgültigen Bilder und Tiefe Stapel Filme wurden unter Verwendung ImageJ durch die Kombination beider Detektionskanäle. Bitte <a href="https://www.jove.com/files/ftp_upload/1692/1692_fig1.jpg">klicken Sie hier,</a> um eine größere Version der Abbildung 1 zu sehen.

<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

Zunehmende Hinweise darauf, dass Kultivierung von Krebszellen in drei Dimensionen genauer rekapituliert die Komplexität der Tumorbiologie. Viele dieser Modelle verwenden rekonstituierte Basalmembran von Tieren, die eine variable Menge von Wachstumsfaktoren und Zytokine, die das Wachstum dieser Zellkultur-Modellen beeinflussen können enthalten abgeleitet. Hier beschreiben wir im Detail die Herstellung und Verwendung von PuraMatrix, ein im Handel erhältliches Selbstmontage Peptid-Gel, das frei von tierischen Materialien und Erregern zu kapseln und zu propagieren Ovarialkarzinom-Zelllinie, OVCAR-5 ist. Wir beginnen mit einer Beschreibung, wie die PuraMatrix vor vorbereiten zu bedienen. Als nächstes zeigen wir, wie man richtig mischen PuraMatrix und Zellsuspension zu den Zellen in das Hydrogel kapseln. Nach der Zugabe von Zellkulturmedien oder Injektion in einer physiologischen Umgebung, die Peptid-Komponente von PuraMatrix schnell selbst montiert in ein 3D-Hydrogel, das im Nanometer-Maßstab faserige Struktur mit einer mittleren Porengröße von 5-200 nm aufweist<sup&gt; 1</sup&gt;. Darüber hinaus zeigen wir, wie man Kulturen in gekapselten PuraMatrix gewachsen zu propagieren. Wenn in PuraMatrix gekapselt, montieren OVCAR-5-Zellen in dreidimensionale acinar Strukturen, die stärker die Morphologie der mikrometastatischen Knötchen in der Klinik als Monolayer in vitro-Modellen beobachtet ähneln. Mit der konfokalen Mikroskopie illustrieren wir das Erscheinungsbild der repräsentativen OVCAR-5-Zellen in PuraMatrix am Tag 1, 3, 5 und 7 nach der Beschichtung verkapselt. Die Verwendung von PuraMatrix zur Kultur Krebszellen sollen zu einem besseren Verständnis der Erkrankung und es uns ermöglichen, Ansprechen auf die Behandlung in mehr klinisch prädiktive Modell-Systemen zu beurteilen.

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

PuraMatrix Encapsulation of Cancer Cells

Dieses Video zeigt, wie Sie kapseln und Kultur Krebszellen in PuraMatrix, ein im Handel erhältliches Selbstmontage Peptid-Gel.

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 Aufrufe.  Harvard Medical School. Dieses Video zeigt, wie Sie kapseln und Kultur Krebszellen in PuraMatrix, ein im Handel erhältliches Selbstmontage Peptid-Gel.

Serie: PuraMatrix Encapsulation of Cancer Cells

Title Cell Encapsulation by Droplets

Titel der Verkapselung von Zellen durch Tröpfchen

Forschung

Biologie

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.

Isolierung und Analyse von hämatopoetischen Stammzellen aus der Plazenta

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.

Cellular Encapsulation in 3D Hydrogele für Tissue Engineering

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.

Isolierung von Stammzellen aus menschlichen Bauchspeicheldrüsenkrebs 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 ...

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.

Isolierung und Kultivierung von Adult epithelialen Stammzellen aus menschlicher Haut

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.

Durchflusszytometrie Reinigung der Maus meiotischen Zellen

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-Kultur von primären humanen Eileiter Epithelzellen

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.

Visualisierung der interstitiellen Zellen von Cajal (ICC) Netzwerk in Mäuse

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.

Isolierung von Immunzellen aus Primärtumoren

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.

Induktion und Analyse von epithelialen zu mesenchymalen Transition

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