テクニカルノート： <ul><li>ゲル化は、塩の添加によって開始されます。したがって、ゲル化が望まれるまでには塩がPuraMatrixに接触しないことが重要ですし、非常に低イオン強度の塩（例えば、&lt;0.05 M）への曝露は、不可逆的なゲル化が発生します。 </li><li>用意されている残りの原液は、彼らは塩と接触されていなければ後日使用することができます。あなたは繰り返し超音波処理を心配する必要はありません。 </li><li>気泡を遠心分離に続いて滅菌チューブにPuraMatrixを小分け削除することができます。 </li><li>簡単にPuraMatrixのベッドを混乱させることができる吸引などのメディアの変化時には十分注意してください。 </li></ul>材料： BD PuraMatrix RAD16のペプチドハイドロゲル1％（カタログ番号354250）：原液混合物の総ペプチドの濃度は10 mg / mlである。 <ul><li>水浴ソニケーターまたはボルテックス</li><li> 96ウェル細胞培養ディッシュ</li><li>細胞培養培地</li><li>滅菌濾過したH 2 O </li><li>滅菌濾過した20％ショ糖</li><li>細胞（全5,000細胞/ウェル）：OVCAR - 5 </li></ul>すべてのステップは、無菌条件下で行う必要があります。 手順： <ol><li>使用前に粘度を減らすために30分間水浴の超音波処理で1％PuraMatrix（RAD16）を超音波で分解する。 </li><li> 1％PuraMatrixのソリューションは現在、将来の実験を簡素化するために滅菌チューブに等分することができます。 <ol class="lalpha"><li>気泡の形成を避け、彼らが形成している場合は単純に5分間1000rpmで（300 × g）でPuraMatrixを含有する滅菌チューブをスピンダウン。 </li><li>原液混合物の総ペプチドの濃度は10 mg / mlである。 </li></ol></li><li> 4ウェルの二組はそれぞれ、2.5 mg / mlの1.25 mg / mlの合計ペプチドの濃度で播種される。 </li><li> 10％ショ糖でBD PuraMatrixの2倍の濃度を達成するために、それぞれ、よく0.2μmの5 mg / mlのと2.5 mg / mlの合計ペプチド濃度まで滅菌濾過した20％ショ糖と13.3％のショ糖で設定された各PuraMatrixを希釈する。 <ol class="lalpha"><li> 20％のショ糖を作るために、次にスクロース10gを、50mlの水にボリュームを希釈する。 </li><li> 13％、10％ショ糖を20％ストック溶液を希釈することにより調製することができる。 <ol class="lroman"><li> 13％ショ糖（各10 mlの）：6.5ミリリットル20％ショ糖+ 3.5 mlの滅菌H 2 O </li><li> 10％ショ糖（各10 mlの）：5.0ミリリットル20％ショ糖+ 5.0 mlの滅菌H 2 O </li></ol></li></ol></li><li>あなたがプレートにしようと井戸の数に基づいてPuraMatrixペプチドのマスターミックスを調製する。 （下記の例を参照してください。）それからマスターミックスの25mlを含む個々のチューブに分注し。 <ol class="lalpha"><li>マスターミックスは、それがメッキ中に細胞の等体積で希釈されるため、合計ペプチドの倍所望の最終濃度に調製してください。 <img src="/files/ftp_upload/1692/1692_fig11.jpg" alt="図1" /></li></ol></li><li>細胞単層培養細胞をトリプシン処理により細胞懸濁液を準備します。 <ol class="lalpha"><li> 0.05％トリプシンEDTA 2mlを各T - 75フラスコに使用することができます。 </li></ol></li><li> 10％の熱不活化ウシ胎児血清を含む細胞培養培地の添加によりトリプシンを中和する（前のステップで使用されるトリプシンのmlあたりのメディアの少なくとも2 mlを使用する。） </li><li>細胞ペレットを作成するために5分間1000rpmで（〜300 × g）で細胞を遠心分離します。 <ol><li> 10パーセント滅菌濾過したショ糖で細胞を再懸濁します。 </li><li>細胞をカウントし、メディアに見られる塩の微量を削除するには、5分間で1000rpm（〜300 × g）で再びスピンダウン。 </li></ol></li><li>ウェル当たりの最終細胞数は、各ウェルに5,000細胞になるように2.0 × 10 5細胞/ mlで10％ショ糖でOVCAR - 5細胞を再懸濁します。 次の手順を迅速に実行する必要があります。 </li><li>上記の回路図に示すようにPuraMatrixのマスターミックス25μlを含む最初の個々のチューブに2.0 × 10 5細胞/ mlの懸濁液25μlを追加します。 <ol class="lalpha"><li>すぐに気泡を導入することなく、チューブに（上下〜5回）ピペッティングにより混和する。 </li><li>その後96ウェルディッシュの適切なウェルに50μlの細胞懸濁液/ PuraMatrix混合物をピペットで。 </li><li>優しくもの側面に150μlの細胞培養培地（RPMI + 10％FBS）をピペッティングでのBD PuraMatrixのゲル化を開始 <img src="/files/ftp_upload/1692/1692_fig12.jpg" alt="図2" /></li></ol></li><li>すべての井戸がメッキされるまで、8で説明した手順を繰り返します。 </li><li> 30分後に非常に穏やかにハイドロゲルのpHを平衡化するために200μlのピペットを使用して、メディアの〜2 / 3を削除。 <ol class="lalpha"><li> これはマトリックスが中断されます、真空吸引器を使用しないでください </li><li> よくなるようにピペットチップを置きますそれは、PuraMatrixのベッドに触れ、ゆっくりとよくからメディアを削除しません。 </li><li> ケアは、PuraMatrixの細胞の培養中に行列を妨害しないように注意してください。 </li></ol></li><li> 非常にゆっくりと PuraMatrix内にカプセル化も含むセルの側面に新たな細胞培養培地150μlを加える。 </li><li>慎重に手順10と11を繰り返すことによって、新鮮な培地で各ウェル毎に48時間の細胞培養培地を変更します。 </li></ol>代表的な結果： <img src="/files/ftp_upload/1692/1692_fig1tmb.jpg" alt="図1" border="0" /> 上記のプロトコルで説明したように図1。OVCAR - 5細胞を封入した。画像は、スペクトラフィジックスDeepSeeのチタンを搭載した倒立オリンパスFV1000顕微鏡を用いて取得されました：サファイアレーザーは750 nmのように調整。イメージングセッションは1日目、3、5と7のポスト - メッキに実施された。 40倍対物レンズ（オリンパスLUMPLFLN 0.8 NA）を浸漬長い作動距離、水がPuraMatrixを通してイメージに使用されていました。三次元ボリュームは、1.47μmの軸方向のステップで画像スタックを取得することにより収集した。二つの非デスキャン検出器は紫（440〜490 nm）と緑（510〜550 nm）のバンドパスフィルタを用いて自家蛍光発光を収集した。最終的な画像と深さのスタックムービーは、両方の検出チャネルを組み合わせることで、ImageJを用いて処理した。してください<a href="https://www.jove.com/files/ftp_upload/1692/1692_fig1.jpg">ここをクリックして</a>図1の拡大バージョンを参照すること。

<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

増加する証拠は、反復する腫瘍生物学の複雑さをより正確に三次元的にその培養癌細胞を示唆している。これらのモデルの多くは、これらの細胞培養モデルの成長に影響を与えることができる成長因子やサイトカインの変数量を含む動物由来の再構成基底膜を利用する。ここで、我々は詳細に説明PuraMatrix、動物由来の材料および卵巣癌細胞株、OVCAR - 5をカプセル化し、伝播する病原体を欠いている市販の自己組み立てペプチドゲルの調製および使用。我々は、使用前にPuraMatrixを準備する方法を説明することから始めます。次に、我々は適切にハイドロゲルに細胞をカプセル化するPuraMatrixと細胞懸濁液を混合する方法を示します。生理的な環境への細胞培養培地または注射を添加すると、PuraMatrixのペプチド成分は急速に自己は、500から200 nmの平均孔径をナノメートルスケールの繊維状の構造を示す3Dハイドロゲルに組み立て<sup&gt; 1</sup&gt;。さらに、我々はカプセル化されたPuraMatrixで育った文化を伝播する方法を示します。 PuraMatrixにカプセル化されたときは、OVCAR - 5細胞は、より密接にin vitroモデル単層よりも診療所で観測された微小結節の形態のようになっていることを三次元腺構造に組み立てる。共焦点顕微鏡を用いて、我々は1日、3、5、および7のポストのメッキにPuraMatrixにカプセル化された代表的なOVCAR - 5細胞の外観を示しています。文化の癌細胞へPuraMatrixの使用は、病気の我々の理解を改善し、より臨床的に予測モデル系における治療効果を評価できるようにする必要があります。

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

PuraMatrix Encapsulation of Cancer Cells

このビデオはPuraMatrix、ペプチドのゲルを組み立てる市販の自己のがん細胞をカプセル化し、文化に方法を示しています。

癌細胞のPuraMatrixのカプセル化

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 ビュー.  Harvard Medical School. このビデオはPuraMatrix、ペプチドのゲルを組み立てる市販の自己のがん細胞をカプセル化し、文化に方法を示しています。

ビデオ: PuraMatrix Encapsulation of Cancer Cells

Title Cell Encapsulation by Droplets

滴タイトルセルのカプセル化

生物学

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.

プラセンタから造血幹細胞の単離と解析

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.

組織工学のための3Dハイドロゲルの細胞のカプセル化

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.

ヒト膵臓癌異種移植片由来の幹細胞の分離

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.

ヒト皮膚から大人上皮幹細胞の単離と培養

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.

マウス減数分裂細胞のフローサイトメトリーの精製

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.

初代ヒト卵管上皮細胞のex vivoでの文化

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.

マウスにおけるカハール（ICC）ネットワークの間質細胞の可視化

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.

原発腫瘍から免疫細胞の分離

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.

間葉転換を誘導し、上皮の分析

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