טכנית הערות: <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Gelation הוא שיזם תוספת של מלחים. לכן, זה קריטי, כי מלחי לא לבוא במגע עם PuraMatrix עד gelation היא הרצויה; חשיפה מלחי כוח יוניים נמוך מאוד (למשל, &lt;0.05 מ &#39;) תגרום gelation בלתי הפיך. </li><li style=";text-align:right;direction:rtl"> פתרונות שאריות מלאי כי הם מוכנים ניתן להשתמש במועד מאוחר יותר, בתנאי שהם לא באים במגע עם מלחים. אתה לא צריך לדאוג sonication לחזור. </li><li style=";text-align:right;direction:rtl"> בועות האוויר ניתן להסיר aliquoting PuraMatrix לתוך צינורות סטרילי ואחריו צנטריפוגה. </li><li style=";text-align:right;direction:rtl"> היזהר מאוד בזמן שינויים מדיה כשאיפה יכול בקלות לשבש את המיטה PuraMatrix. </li></ul> חומרים: BD PuraMatrix פפטיד RAD16 Hydrogel-1% (Cat # 354250): ריכוז הפפטיד הכולל של התערובת חי היא 10 מ&quot;ג / מ&quot;ל. <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> אמבט מים Sonicator או וורטקס </li><li style=";text-align:right;direction:rtl"> 96, גם בתא תרבות צלחת </li><li style=";text-align:right;direction:rtl"> תרבית תאים מדיה </li><li style=";text-align:right;direction:rtl"> סינון סטרילי H 2 O </li><li style=";text-align:right;direction:rtl"> סטרילי סוכרוז 20% מסוננים </li><li style=";text-align:right;direction:rtl"> תאים (כל התאים ב 5000 / טוב): OVCAR-5 </li></ul> כל הצעדים חייבים להתבצע בתנאים aseptic. נוהל: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Sonicate PuraMatrix 1% (RAD16) ב sonicator מים באמבטיה במשך 30 דקות כדי להפחית את צמיגות לפני השימוש. </li><li style=";text-align:right;direction:rtl"> הפתרון PuraMatrix 1% כעת ניתן aliquoted לתוך צינורות סטרילי כדי לפשט ניסויים עתידיים. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הימנע היווצרות של בועות אוויר, אם הם יוצרים פשוט ספין את צינורות סטרילי המכיל PuraMatrix ב 1000 סל&quot;ד (300 XG) במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> ריכוז הפפטיד הכולל של התערובת חי היא 10 מ&quot;ג / מ&quot;ל. </li></ol></li><li style=";text-align:right;direction:rtl"> שני סטים של 4 בארות יהיה מצופה ב 2.5 מ&quot;ג / מ&quot;ל ​​ו - 1.25 מ&quot;ג / מ&quot;ל ​​פפטיד ריכוז מוחלט, בהתאמה. </li><li style=";text-align:right;direction:rtl"> לדלל את PuraMatrix בכל להגדיר היטב עם 0.2 מיקרומטר סטרילי סוכרוז 20% מסונן סוכרוז 13.3% לריכוזים פפטיד סך של 5 מ&quot;ג / מ&quot;ל ​​ו - 2.5 מ&quot;ג / מ&quot;ל, בהתאמה כדי להשיג ריכוז של 2x BD PuraMatrix ב סוכרוז 10%. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> To Make סוכרוז 20%, לדלל 10 גרם של סוכרוז, אז נפח 50 מ&quot;ל מים. </li><li style=";text-align:right;direction:rtl"> 13% סוכרוז ו 10% יכול להיות מוכן על ידי דילול פתרון המניה 20%. <ol class="lroman" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> סוכרוז 13% (עבור כל 10 מ&quot;ל): 6.5 מ&quot;ל סוכרוז 20% + 3.5 מ&quot;ל סטרילי H 2 O. </li><li style=";text-align:right;direction:rtl"> 10% סוכרוז (עבור כל 10 מ&quot;ל): 5.0 מ&quot;ל סוכרוז 20% + 5.0 מ&quot;ל סטרילי H 2 O. </li></ol></li></ol></li><li style=";text-align:right;direction:rtl"> הכינו תערובת אמן PuraMatrix הפפטיד מבוסס על מספר בארות אתה מתכוון צלחת. (ראו דוגמה להלן). Aliquot ואז לתוך צינורות בודדים המכיל 25 מ&quot;ל של תערובת האב. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לערבב האב צריך להיות מוכן בריכוז פעמיים הסופית הרצויה של הפפטיד הכולל כפי שהוא יהיה מדולל עם נפח שווה של תאים במהלך ציפוי. <img src="/files/ftp_upload/1692/1692_fig11.jpg" alt="דמות 1" /></li></ol></li><li style=";text-align:right;direction:rtl"> הכן השעיות התא על ידי trypsinizing monolayer תא בתרביות תאים. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 2 מ&quot;ל של 0.05% טריפסין EDTA יכול לשמש Flask T-75 כל אחד. </li></ol></li><li style=";text-align:right;direction:rtl"> לנטרל את טריפסין על ידי תוספת של תרבות התקשורת התא המכיל 10% נסיוב מומת חום שור העובר (להשתמש לפחות 2 מ&quot;ל של התקשורת לכל מ&quot;ל של טריפסין משמש בשלב הקודם). </li><li style=";text-align:right;direction:rtl"> צנטריפוגה התאים ב 1000 סל&quot;ד (~ 300 XG) במשך 5 דקות כדי ליצור תא גלולה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Resuspend התאים סוכרוז 10% סינון סטרילי. </li><li style=";text-align:right;direction:rtl"> ספירת התאים ספין שוב ב 1000 סל&quot;ד (~ 300 XG) במשך 5 דקות להסיר כמויות זעירות של מלח נמצא בתקשורת. </li></ol></li><li style=";text-align:right;direction:rtl"> Resuspend OVCAR-5 תאים סוכרוז 10% ב 2.0 x 10 5 תאים / מ&quot;ל כך לספור התא הסופי לכל טוב יהיה טוב 5000 תאים כל אחד. השלבים הבאים יש לבצע במהירות. </li><li style=";text-align:right;direction:rtl"> הוסף 25 μl של 10 x 2.0 5 תאים / מ&quot;ל השעיה אל הצינור היחיד first המכיל 25 μl של תמהיל אמן PuraMatrix כפי שמוצג סכמטית לעיל. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לערבב במהירות על ידי pipetting (~ 5 פעמים הלוך ושוב) בצינור ללא החדרת בועות אוויר. </li><li style=";text-align:right;direction:rtl"> ואז פיפטה 50 תערובת μl השעיה / PuraMatrix התא לתוך הבאר המתאים מנה 96 באר. </li><li style=";text-align:right;direction:rtl"> ליזום gelation של PuraMatrix BD ידי בעדינות pipetting 150 μl תרבית תאים מדיה (RPMI + 10% FBS) לצד הבאר <img src="/files/ftp_upload/1692/1692_fig12.jpg" alt="דמות 2" /></li></ol></li><li style=";text-align:right;direction:rtl"> חזור על הפעולות המתוארות 8 עד שכל בארות כבר מצופה. </li><li style=";text-align:right;direction:rtl"> לאחר 30 דקות להסיר בעדינות רבה ~ 2 / 3 של התקשורת באמצעות פיפטה 200 μl לאזן את רמת החומציות של הידרוג. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> אל תשתמש ASPIRATOR אבק כמו זה ישבש את MATRIX </li><li style=";text-align:right;direction:rtl"> הניחו את קצה פיפטה את הבאר כךזה לא נוגע במיטה PuraMatrix בעדינות להסיר מדיה מן הבאר. </li><li style=";text-align:right;direction:rtl"> יש להקפיד לא לשבש את מטריקס במהלך התרבות התאים PuraMatrix. </li></ol></li><li style=";text-align:right;direction:rtl"> בעדינות להוסיף 150 μl של התקשורת בתרבות טרי תא לצד התאים המכילים גם הגלום PuraMatrix. </li><li style=";text-align:right;direction:rtl"> שינוי התרבות תאים התקשורת בכל טוב כל 48 שעות עם התקשורת בתרבות טרי בקפידה על ידי חזרה על שלבים 10 ו -11. </li></ol> נציג תוצאות: <img src="/files/ftp_upload/1692/1692_fig1tmb.jpg" alt="דמות 1" border="0" /> באיור 1. OVCAR-5 תאים היו ארוזים כמתואר בפרוטוקול לעיל. תמונות נרכשו באמצעות הפוכה אולימפוס FV1000 מיקרוסקופ המצויד Spectra-Physics DeepSee טי: ספיר לייזר מכוון 750 ננומטר. הפעלות הדמיה שנערכו בימים 1, 3, 5 ו - 7 שלאחר ציפוי. מרחק עבודה ארוך, טובלים במים אובייקטיבי 40X (אולימפוס LUMPLFLN 0.8 NA) שימש תמונה דרך PuraMatrix. תלת ממדי כרכים נאספו באמצעות רכישת ערימות התמונה צעדים 1.47 מיקרומטר צירית. שני אי - descanned גלאי אסף את פליטת autofluorescence באמצעות סגול (440-490 ננומטר) וירוק (510-550 ננומטר) מסננים bandpass. התמונות הסופית וסרטים מחסנית עומק עובדו באמצעות ImageJ על ידי שילוב שני ערוצי גילוי. אנא <a href="https://www.jove.com/files/ftp_upload/1692/1692_fig1.jpg">לחץ כאן</a> כדי לראות גרסה גדולה יותר של דמות 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

הראיות עולה כי הגדלת תאים סרטניים culturing בשלושה ממדים משחזר בצורה מדויקת יותר את המורכבות של ביולוגיה הגידול. רבים מן המודלים האלה לנצל קרום במרתף מחדש שמקורם בבעלי חיים אשר מכילים כמות משתנה של גורמי גדילה ציטוקינים שיכולים להשפיע על הצמיחה של המודלים האלה תרבית תאים. כאן אנו מתארים בפרוטרוט את ההכנה והשימוש PuraMatrix, זמין מסחרית הרכבה עצמית פפטיד ג&#39;ל כי הוא נטול חומרים שמקורם בבעלי חיים ומזיקים כדי לתמצת להפיץ את סרטן השחלות התא קו, OVCAR-5. נתחיל המתאר כיצד להכין את PuraMatrix לפני השימוש. בשלב הבא, אנחנו מדגימים איך שצריך לערבב את PuraMatrix ההשעיה התא כדי לתמצת את התאים הידרוג. עם תוספת של תרבות המדיה הנייד או הזרקה לסביבה פיזיולוגית, רכיב של הפפטיד PuraMatrix במהירות עצמי מרכיב לתוך הידרוג 3D שמספקת בקנה מידה ננומטרי מבנה סיבי עם גודל נקבוביות ממוצע של 500-200 ננומטר<sup&gt; 1</sup&gt;. בנוסף, אנו מדגימים כיצד להפיץ תרבויות גדל PuraMatrix כמוס. כאשר הגלום PuraMatrix, OVCAR-5 תאים להרכיב לתוך שלושה מבנים acinar ממדי דומים יותר את המורפולוגיה של גושים micrometastatic שנצפו במרפאה מאשר monolayer במודלים חוץ גופית. באמצעות מיקרוסקופיה confocal לנו להמחיש את המראה של נציג OVCAR-5 תאים הגלום PuraMatrix על 1 יום, 3, 5, 7 ו ציפוי הודעה. השימוש PuraMatrix לתאי סרטן תרבות צריך לשפר את הבנתנו של המחלה מאפשר לנו להעריך את התגובה לטיפול במערכות מודל החיזוי יותר מבחינה קלינית.

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

PuraMatrix Encapsulation of Cancer Cells

סרט הווידאו הזה מדגים כיצד לתמצת תאים סרטניים תרבות PuraMatrix, עצמי זמינים מסחרית להרכבת ג&#39;ל פפטיד.

PuraMatrix Encapsulation של תאים סרטניים

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. סרט הווידאו הזה מדגים כיצד לתמצת תאים סרטניים תרבות PuraMatrix, עצמי זמינים מסחרית להרכבת ג'ל פפטיד.

Video: PuraMatrix Encapsulation of Cancer Cells

Title Cell Encapsulation by Droplets

תא כותרת Encapsulation על ידי טיפות

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 נייד ב הידרוג 3D עבור הנדסת רקמות

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.

גזירת Trophoblast עכבר בתאי גזע Blastocysts

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.

בידוד של בתאי גזע xenografts סרטן הלבלב האנושי

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.

בידוד תרבות למבוגרים גזע תאים אפיתל של העור האנושי

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.

Cytometry זרימה טיהור תאים עכבר 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 Vivo התרבות של האדם תאים ראשוניים החצוצרה אפיתל

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.

ויזואליזציה של תאים ביניים של רשת (ICC) קחאל ב עכברים

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.

בידוד של תאים חיסוניים של גידולים ראשוניים

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.

אינדוקציה וניתוח של אפיתל לmesenchymal מעבר

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