ملاحظات فنية : <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يبدأ تهليم من خلال إضافة الأملاح. لذا ، فمن الأهمية بمكان أن لا أملاح تأتي في اتصال مع PuraMatrix هو المطلوب حتى تهليم ؛ التعرض للأملاح أيونية قوة منخفضة جدا (على سبيل المثال ، &lt;0.05 م) سوف يتسبب تهليم لا رجعة فيه. </li><li style=";text-align:right;direction:rtl"> ويمكن استخدام الحلول الأسهم المتبقية التي يتم إعدادها في وقت لاحق شريطة ألا يكونوا قد تأتي في اتصال مع الأملاح. كنت لا داعي للقلق بشأن صوتنة تكرار. </li><li style=";text-align:right;direction:rtl"> ويمكن إزالة فقاعات الهواء aliquoting PuraMatrix في أنابيب معقمة تليها الطرد المركزي. </li><li style=";text-align:right;direction:rtl"> نكون حذرين للغاية خلال التغييرات وسائل الاعلام كما يمكن أن يحدث خللا تطلع بسهولة PuraMatrix السرير. </li></ul> المواد : دينار بحريني الببتيد RAD16 PuraMatrix هيدروجيل - 1 ٪ (القط # 354250) : إن تركيز الببتيد الإجمالية للخليط مخفف هو 10 ملغ / مل. <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> المياه Sonicator حمام أو دوامة </li><li style=";text-align:right;direction:rtl"> 96 - جيدا ثقافة خلية طبق </li><li style=";text-align:right;direction:rtl"> خلية ثقافة وسائل الإعلام </li><li style=";text-align:right;direction:rtl"> معقمة المصفاة H 2 O </li><li style=";text-align:right;direction:rtl"> معقمة المصفاة السكروز 20 ٪ </li><li style=";text-align:right;direction:rtl"> خلايا (جميع الخلايا في 5000 / إضافة) : OVCAR - 5 </li></ul> يجب تنفيذ جميع الخطوات تحت ظروف معقمة. الإجراء : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يصوتن للPuraMatrix 1 ٪ (RAD16) في المياه sonicator الحمام لمدة 30 دقيقة لتقليل اللزوجة قبل الاستخدام. </li><li style=";text-align:right;direction:rtl"> ويمكن الآن حل PuraMatrix 1 ٪ يكون في أنابيب معقمة aliquoted لتبسيط التجارب في المستقبل. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تجنب تكون فقاعات الهواء ، وإذا كانت تدور ببساطة شكل أسفل أنابيب معقمة تحتوي PuraMatrix دورة في الدقيقة 1000 (300 x ج) لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> تركيز الببتيد الإجمالية للخليط مخفف هو 10 ملغ / مل. </li></ol></li><li style=";text-align:right;direction:rtl"> وسيتم مطلي مجموعتين من 4 آبار بنسبة 2.5 ملغ / مل و 1.25 ملغ / مل تركيز الببتيد المجموع ، على التوالي. </li><li style=";text-align:right;direction:rtl"> تمييع PuraMatrix في كل مجموعة بشكل جيد مع 0.2 ميكرون معقمة تصفيتها السكروز 20 ٪ و 13.3 ٪ سكروز لتركيزات الببتيد مجموعه 5 مل / ملغ و 2.5 ملغ / مل على التوالي لتحقيق تركيز 2X من PuraMatrix دينار بحريني في السكروز 10 ٪. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جعل السكروز 20 ٪ ، 10 غراما من تمييع السكروز ، ثم وحدة التخزين إلى 50 مل من الماء. </li><li style=";text-align:right;direction:rtl"> يمكن إعداد 13 ٪ و 10 ٪ سكروز تمييع الحل الأسهم 20 ٪. <ol class="lroman" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> السكروز 13 ٪ (مقابل كل مل 10) : 6.5 مل السكروز 20 ٪ + 3.5 مل العقيمة H 2 O. </li><li style=";text-align:right;direction:rtl"> 10 ٪ سكروز (لكل 10 مل) : 5.0 مل السكروز 20 ٪ + 5.0 مل العقيمة H 2 O. </li></ol></li></ol></li><li style=";text-align:right;direction:rtl"> تحضير مزيج سيد الببتيد PuraMatrix استنادا إلى عدد من الآبار التي تنوي لوحة. (انظر المثال أدناه). ثم قسامة في أنابيب الفردية التي تحتوي على 25 مل من مزيج الرئيسي. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وينبغي إعداد هذا المزيج الرئيسي في ضعف التركيز النهائي المطلوب من مجموع الببتيد كما سيتم تخفيفه مع حجم مساو من خلايا أثناء الطلاء. <img src="/files/ftp_upload/1692/1692_fig11.jpg" alt="الشكل 1" /></li></ol></li><li style=";text-align:right;direction:rtl"> إعداد تعليق خلية خلية خلية trypsinizing الثقافات أحادي الطبقة. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ويمكن استخدام 2 مل من 0.05 ٪ التربسين EDTA لكل قارورة T - 75. </li></ol></li><li style=";text-align:right;direction:rtl"> تحييد التربسين من قبل وسائل الإعلام بالإضافة إلى زراعة الخلايا التي تحتوي على 10 ٪ الحرارة المعطل الجنين المصل البقري (استخدم ما لا يقل عن 2 مل من وسائل الإعلام لكل مل من التربسين المستخدمة في الخطوة السابقة). </li><li style=";text-align:right;direction:rtl"> الطرد المركزي الخلايا الدقيقة في 1000 (~ 300 x ج) لمدة 5 دقائق لإنشاء خلية بيليه. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Resuspend الخلايا في السكروز 10 ٪ العقيمة التي تمت تصفيتها. </li><li style=";text-align:right;direction:rtl"> عدد الخلايا وتدور مرة أخرى في أسفل دورة في الدقيقة 1000 (~ 300 x ج) لمدة 5 دقائق لإزالة كميات ضئيلة من الملح وجدت في وسائل الإعلام. </li></ol></li><li style=";text-align:right;direction:rtl"> Resuspend الخلايا OVCAR - 5 في السكروز 10 ٪ في الدورة العاشرة لل2.0 10 5 خلية / مل بحيث الفرز النهائي في الخلية جيدا وسوف 5000 الخلايا في كل بئر. يجب أن يتم تنفيذ الخطوات التالية بسرعة. </li><li style=";text-align:right;direction:rtl"> إضافة ميكرولتر من 25 س 2.0 10 5 خلية / مل تعليق لأنبوب الفرد الأول الذي يحتوي 25 ميكروليتر من مزيج سيد PuraMatrix كما هو موضح في التخطيطي أعلاه. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> المزيج بسرعة pipetting (~ 5 مرات صعودا وهبوطا) في أنبوب دون إدخال فقاعات الهواء. </li><li style=";text-align:right;direction:rtl"> ماصة ثم 50 ميكرولتر الخلية تعليق / PuraMatrix الخليط المناسب من البئر 96 طبق بشكل جيد. </li><li style=";text-align:right;direction:rtl"> بدء تهليم من PuraMatrix دينار بحريني من قبل pipetting بلطف ميكرولتر 150 خلية ثقافة وسائل الإعلام (+ 10 ٪ RPMI FBS) إلى جانب البئر <img src="/files/ftp_upload/1692/1692_fig12.jpg" alt="الشكل 2" /></li></ol></li><li style=";text-align:right;direction:rtl"> كرر الخطوات المذكورة في 8 حتى تم مطلي جميع الآبار. </li><li style=";text-align:right;direction:rtl"> بعد 30 دقيقة جدا إزالة برفق ~ 2 / 3 من وسائل الإعلام باستخدام ماصة 200 ميكرولتر للتوازن الرقم الهيدروجيني للهيدروجيل. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لا تستخدم الشافطة الكهربائية حيث سيؤدي ذلك إلى تعطيل ماتركس </li><li style=";text-align:right;direction:rtl"> مكان غيض ماصة في البئر بحيثلا تلمس السرير برفق PuraMatrix وإزالة وسائل الاعلام من البئر. </li><li style=";text-align:right;direction:rtl"> ينبغي الحرص على عدم تعطيل المصفوفة خلال ثقافة الخلايا في PuraMatrix. </li></ol></li><li style=";text-align:right;direction:rtl"> إضافة جدا برفق 150 ميكرولتر من خلية ثقافة جديدة وسائل الاعلام الى جانب الخلايا التي تحتوي كذلك مغلفة في PuraMatrix. </li><li style=";text-align:right;direction:rtl"> تغيير ثقافة وسائل الإعلام في كل خلية ساعة كل 48 جيدا مع وسائل الاعلام ثقافة جديدة بتكرار الخطوات بعناية 10 و 11. </li></ol> ممثل النتائج : <img src="/files/ftp_upload/1692/1692_fig1tmb.jpg" alt="الشكل 1" border="0" /> الشكل 1. كانت مغلفة OVCAR - 5 الخلايا كما هو موضح في البروتوكول أعلاه. تم الحصول على الصور باستخدام مجهر مقلوب أوليمبوس FV1000 مجهزة تي DeepSee الأطياف ، فيزياء : الياقوت الليزر ضبطها إلى 750 نانومتر. وأجريت دورات التصوير في أيام 1 و 3 و 5 و 7 بعد الطلاء. واستخدمت مسافة طويلة في العمل ، والمياه غمس الهدف 40X (0.8 أوليمبوس LUMPLFLN NA) لصورة من خلال PuraMatrix. جمعت ثلاثي الأبعاد من خلال الحصول على كميات مداخن الصورة في الخطوات 1.47 ميكرون المحوري. جمعت اثنين من غير descanned كشف عن انبعاث تألق ذاتي باستخدام البنفسجي (440-490 نانومتر) والأخضر (510-550 نانومتر) مرشحات ممر الموجة. تمت معالجة الصور والأفلام النهائي عمق المكدس باستخدام ImageJ عن طريق الجمع بين كل من قنوات الكشف. الرجاء <a href="https://www.jove.com/files/ftp_upload/1692/1692_fig1.jpg">انقر هنا</a> لرؤية نسخة أكبر من الرقم 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

أدلة متزايدة تشير إلى أن زراعة الخلايا السرطانية في ثلاثة أبعاد أكثر دقة يلخص تعقيد البيولوجيا الورم. كثير من هذه النماذج تستخدم تشكيل الغشاء القاعدي المستمدة من الحيوانات التي تحتوي على كميات متفاوتة من عوامل النمو والسيتوكينات التي يمكن أن تؤثر على نمو هذه النماذج ثقافة الخلية. هنا ، نحن تصف بالتفصيل إعداد واستخدام PuraMatrix ، وهي متوفرة تجاريا هلام الذاتي تجميع الببتيد التي تخلو من الحيوانات المستمدة من المواد والعوامل الممرضة لتغليف ونشر الخلايا السرطانية خط المبيض ، OVCAR - 5. نبدأ تصف كيفية إعداد PuraMatrix قبل الاستخدام. المقبل ، ونحن لشرح كيفية مزج صحيح PuraMatrix وتعليق خلية لتغليف الخلايا في هيدروجيل. بالإضافة إلى ذلك على وسائل الإعلام أو الثقافة حقن الخلايا في بيئة الفسيولوجية ، المكون من الببتيد PuraMatrix بسرعة النفس تجمع في هيدروجيل 3D أن يسلك الليفية مقياس النانومتر هيكل مع متوسط ​​حجم المسام من 500-200 نانومتر<sup&gt; 1</sup&gt;. بالإضافة إلى ذلك ، نحن لشرح كيفية نشر الثقافات التي تزرع في PuraMatrix مغلفة. عندما مغلفة في PuraMatrix ، OVCAR - 5 خلايا التجمع في ثلاثة هياكل الابعاد عنيبية أن أكثر شبها مورفولوجيا العقيدات micrometastatic الملاحظة في العيادة في نماذج من المونولاير المختبر. باستخدام المجهر مبائر نوضح ظهور الخلايا ممثل - 5 OVCAR PuraMatrix مغلفة في يوم 1 و 3 و 5 و 7 الطلاء آخر. وينبغي استخدام PuraMatrix على خلايا سرطانية في ثقافة تحسين فهمنا لهذا المرض وتسمح لنا لتقييم الاستجابة للعلاج في النظم نموذج أكثر التنبؤية سريريا.

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

PuraMatrix Encapsulation of Cancer Cells

هذا الفيديو يوضح كيفية تغليف الخلايا السرطانية والثقافة في PuraMatrix ، وهي متوفرة تجاريا الذاتي تجميع جل الببتيد.

PuraMatrix تغليف الخلايا السرطانية

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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Biology

16.6K Views.  Harvard Medical School. هذا الفيديو يوضح كيفية تغليف الخلايا السرطانية والثقافة في PuraMatrix ، وهي متوفرة تجاريا الذاتي تجميع جل الببتيد.

Video: PuraMatrix Encapsulation of Cancer Cells

Title Cell Encapsulation by Droplets

عنوان تغليف الخلية عن طريق الرذاذ

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.

تغليف الخلوية في الهلاميات المائية للهندسة الأنسجة 3D

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.

اشتقاق الخلايا الجذعية من الأرومة الغاذية 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.

عزل الخلايا الجذعية من الإنسان Xenografts سرطان البنكرياس

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.

العزلة وثقافة الكبار الخلايا الجذعية الظهارية من الجلد البشري

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.

تدفق تنقية الخلوي للخلايا الفأر الانتصافي

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.

الثقافة السابقين فيفو من خلايا الإنسان الأولية قناة فالوب طلائي

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.

تصور من خلايا خلالية لشبكة (ICC) في الفئران كاجال

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.

عزل الخلايا المناعية من الأورام الأولية

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.

الاستقراء وتحليل الظهارية الوسيطة الانتقالية ل

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