테크니컬 노트 : <ul><li> 겔화는 소금의 추가에 의해 시작됩니다. 따라서 겔화가 원하는 때까지는 아무 소금이 PuraMatrix와 접촉하지 않는 것이 중요합니다, 매우 낮은 이온 강도의 염 (예를 들어, &lt;0.05 M)에 대한 노출은 돌이킬 수 겔화의 원인이됩니다. </li><li> 준비가되어 남은 주식 솔루션들은 소금​​과 접촉하지 않은 제공 나중에 사용할 수 있습니다. 당신은 반복 sonication 걱정할 필요가 없습니다. </li><li> 공기 방울은 원심 분리에 의해 다음 멸균 튜브에 PuraMatrix를 aliquoting 제거할 수 있습니다. </li><li> 흡인 쉽게 PuraMatrix 침대를 중단 수있는 미디어를 변경하는 동안 매우주의하십시오. </li></ul> 자료 : BD PuraMatrix RAD16의 펩타이드 히드로겔 - 1 % (고양이 # 354250) : undiluted 혼합물의 총 펩타이드의 농도는 10 MG / ML입니다. <ul><li> 워터 목욕 Sonicator 또는 소용돌이 </li><li> 96 - 웰 세포 배양 접시 </li><li> 세포 배양 미디어 </li><li> 멸균 필터링 H 2 O </li><li> 무균 필터링된 20 % 자당 </li><li> 셀 (전체 5,000 세포에 / 잘) OVCAR - 5 </li></ul> 모든 단계는 무균 조건 하에서 수행되어야합니다. 절차 : <ol><li> 사용하기 전에 점도를 줄이기 위해 30 분 물 목욕 sonicator의 1 % PuraMatrix (RAD16)를 Sonicate. </li><li> 1 % PuraMatrix 솔루션은 이제 미래의 실험을 단순화하기 위해 멸균 튜브에 aliquoted 수 있습니다. <ol class="lalpha"><li> 공기 거품의 형성을 피하, 그들이 양식을 경우에는 간단하게 5 분 1,000 RPM (300 XG)에서 PuraMatrix를 포함하는 멸균 튜브를 스핀 다운. </li><li> undiluted 혼합물의 총 펩타이드의 농도는 10 MG / ML입니다. </li></ol></li><li> 4 웰스 두 세트는 각각 2.5 MG / ML 및 1.25 MG / ML 총 펩타이드 농도에서 도금한다. </li><li> 10 %의 자당의 BD PuraMatrix의 2 배 농도를 달성하기 위해 각각 잘 0.2 μm의 5 MG / ML 2.5 MG / ML 총 펩타이드 농도로 멸균 필터링 20 % 자당 및 13.3 %의 자당 설정된 각 PuraMatrix을 희석. <ol class="lalpha"><li> 20 %의 자당을 만들려면 다음 자당의 10g, 볼륨 50 ML 물로 희석. </li><li> 13 %, 10 %의 자당은 20 % 주식 솔루션을 diluting으로 준비하실 수 있습니다. <ol class="lroman"><li> 13 % 자당 (매 10 ML 용) : 6.5 ML 20 %의 자당 + 3.5 ML 살균 H 2 O. </li><li> 10% 자당 (매 10 ML 용) : 5.0 ML 20 %의 자당 + 5.0 ML 살균 H 2 O. </li></ol></li></ol></li><li> 당신이 접시하고자하는 우물의 숫자에 따라 PuraMatrix의 펩타이드의 마스터 믹스를 준비합니다. (아래 예제 참조). 그리고 마스터 믹스 25 ML을 포함하는 각각의 튜브로 나누어지는. <ol class="lalpha"><li> 그것이 도금하는 동안 세포의 동일한 볼륨에 희석 수로 마스터 믹스는 총 펩타이드의 두배가 원하는 최종 농도에서 준비되어야합니다. <img src="/files/ftp_upload/1692/1692_fig11.jpg" alt="그림 1" /></li></ol></li><li> 세포 monolayer 셀 문화를 trypsinizing하여 셀 suspensions를 준비합니다. <ol class="lalpha"><li> 0.05 % 트립신 EDTA (에틸렌 다이아 민 테트라 초산) 2 ML 각 T - 75 플래 사용할 수 있습니다. </li></ol></li><li> 10 %의 열 inactivated 태아 소 혈청을 (이전 단계에서 사용되는 트립신의 ML 당 미디어 최소한 2 ML)을 사용합니다. 포함된 세포 배양 매체 이외에 의해 트립신을 중화 </li><li> 세포 펠렛을 만드는 5 분 1,000 RPM (~ 300 XG)에서 세포를 원심 분리기. <ol><li> 10% 멸균 필터링 자당의 세포를 Resuspend. </li><li> 세포를 카운트하고 미디어에있는 소금 성분을 제거하는 5 분 1,000 RPM (~ 300 XG)에서 다시 다운 스핀. </li></ol></li><li> 물론 당 최종 셀 개수는 각 우물에 5,000 세포됩니다 5 전지 / ML 있도록 2.0 X 10 10 %의 자당의 OVCAR - 5 세포를 Resuspend. 다음 단계는 급속하게 수행하여야한다. </li><li> 로 위의 설계도에 표시된 PuraMatrix의 마스터 믹스 25 μl를 포함하는 첫 번째 개인 튜브에 2.0 X 10 5 셀 / ML 정지 25 μl를 추가합니다. <ol class="lalpha"><li> 신속하게 공기 방울을 도입하지 않고 튜브 pipetting (위, 아래로 ~ 5 배)로 섞는다. </li><li> 다음 96 잘 요리의 적절한 우물에 50 μl 세포 현탁액 / PuraMatrix 혼합물을 피펫. </li><li> 부드럽게 잘 측면 150 μl 세포 배양 미디어 (RPMI + 10% FBS)을 pipetting하여 BD의 PuraMatrix의 겔화을 시작 <img src="/files/ftp_upload/1692/1692_fig12.jpg" alt="그림 2" /></li></ol></li><li> 모든 우물이 도금 때까지 8 설명한 단계를 반복합니다. </li><li> 30 분 후에 아주 부드럽게 히드로겔의 산도를 평형에 200 μl 피펫을 사용하여 미디어의 ~ 2 / 3 제거합니다. <ol class="lalpha"><li> 이것은 행렬을 방해하므로 진공 흡인기를 사용하지 마십시오 </li><li> 그래서 우물에 피펫 팁을 플레이스그것은 PuraMatrix 침대를 터치하고 부드럽게 우물에서 미디어를 제거하지 않습니다. </li><li> 치료는 PuraMatrix에서 세포의 문화 중에 매트릭스를 방해하지 않도록 주의해야한다. </li></ol></li><li> 아주 부드럽게 PuraMatrix에 캡슐 잘 들어있는 세포의 측면으로 신선한 세포 배양 매체 150 μl를 추가합니다. </li><li> 신중하게 단계 10 및 11를 반복하여 새로운 문화 매체와 각 잘마다 48 시간 이내에 세포 배양 매체를 변경합니다. </li></ol> 대표 결과 : <img src="/files/ftp_upload/1692/1692_fig1tmb.jpg" alt="그림 1" border="0" /> 그림은 1. OVCAR - 5 세포는 위의 프로토콜에서 설명한대로 캡슐되었습니다. 사파이어 레이저 750 nm의로 조정 : 이미지는 스펙트럼 - 물리 DeepSee 티 갖춘 거꾸로 올림푸스 FV1000 현미경을 사용하여 인수했다. 이미징 세션은 일 1, 3, 5 7 이후 도금을 실시했다. 40x 목적을 찍기 긴 작동 거리, 물 (올림푸스 LUMPLFLN 0.8 NA)는 PuraMatrix을 통해 이미지로 사용되었습니다. 입체적인 볼륨이 1.47 μm의 축 단계에서 이미지 스택을 인수함으로써 수집되었다. 투 비 descanned 감지기 바이올렛 (440-490 nm의)과 녹색 (510-550 NM) 대역 통과 필터를 사용하여 autofluorescence 방출을 수집했습니다. 최종 이미지와 깊이 스택 영화는 모두 탐지 채널을 결합하여 ImageJ를 사용하여 처리되었습니다. 하시기 바랍니다 <a href="https://www.jove.com/files/ftp_upload/1692/1692_fig1.jpg">여기를 클릭</a> 그림 1의 더 큰 버전을 볼 수 있습니다.

<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

증가 증거 recapitulates 종양 생물학의 복잡보다 정확하게 3 차원에 해당 culturing의 암 세포를 제안합니다. 이러한 모델의 대부분은 이러한 세포 배양 모델의 성장에 영향을 미칠 수 성장 요인과 크린 시토킨의 변수 금액을 포함하는 동물에서 파생된 재구성 지하 막을 활용한다. 여기서는 자세하게 설명 PuraMatrix, 동물 파생 자료와 난소암 세포 라인, OVCAR - 5 캡슐하고 전파하는 병원균의 찾아볼 수있는 상용 자기 조립 펩티드 젤의 준비와 사용합니다. 우리는 사용하기 전에 PuraMatrix를 준비하는 방법을 설명하여 시작합니다. 다음, 우리는 제대로 히드로겔에있는 세포를 캡슐로 PuraMatrix와 세포 현탁액을 혼합하는 방법을 보여줍니다. 생리적 환경으로 세포 배양 미디어 또는 분사의 추가시 PuraMatrix의 펩타이드 성분이 빠르게 자체 5-200 NM의 평균 기공 크기가 나노미터 규모의 섬유 구조를 전시하고 3D 히드로겔로 조립<sup&gt; 1</sup&gt;. 또한, 우리는 캡슐 PuraMatrix 재배 문화를 전파하는 방법을 보여줍니다. PuraMatrix에서 캡슐 때, OVCAR - 5 세포는보다 밀접하게 체외 모델에서 monolayer보다 병원에서 관찰 micrometastatic nodules의 형태와 유사한 것을 입체 acinar 구조로 조립. 공촛점 현미경을 사용하여 우리는 하루에 1, 3, 5, 7 게시 도금에 PuraMatrix에 캡슐 대표 OVCAR - 5 세포의 모양을 보여줍니다. 문화 암세포에 PuraMatrix의 사용은 질병의 우리의 이해를 개선하고 우리는 더 많은 임상 예측 모델 시스템의 치료 반응을 평가하기 위해 허용해야합니다.

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

PuraMatrix Encapsulation of Cancer Cells

이 동영상은 PuraMatrix, 펩타이드 젤 조립 시판 자체 캡슐 문화 암세포하는 방법을 보여줍니다.

암세포의 PuraMatrix의 캡슐화

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 조회수.  Harvard Medical School. 이 동영상은 PuraMatrix, 펩타이드 젤 조립 시판 자체 캡슐 문화 암세포하는 방법을 보여줍니다.

비디오: PuraMatrix Encapsulation of Cancer Cells

Title Cell Encapsulation by Droplets

흩뿌려진의 제목 셀 캡슐화

연구

생물학

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.

태반에서 조혈 줄기 세포의 분리 및 분석

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.

조직 공학을위한 3D Hydrogels에서 셀룰러 봉지

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.

인간 췌장암의 Xenografts에서 줄기 세포의 분리

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.

사람의 피부에서 성인 상피 줄기 세포의 분리 및 문화

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.

마우스 Meiotic 셀 유동세포계측법의 정제

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.

기본 인간 난관 상피 세포의 전의 VIVO 문화

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.

마우스의 Cajal (ICC) 네트워크의 중간 세포의 시각화

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.

일차 종양에서 면역 세포의 분리

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.

간엽 전환을 유도하고 상피의 분석

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