<ol>
	<li>Crow, J., Amso, N. N., Lewin, J., Shaw, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+ultrastructure+of+fallopian+tube+epithelium+at+different+stages+of+the+menstrual+cycle+and+menopause.">Morphology and ultrastructure of fallopian tube epithelium at different stages of the menstrual cycle and menopause.</a> Hum Reprod. 9 (12), 2224-2233 (1994).</li><li>Patek, E., Nilsson, L., Johannisson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+electron+microscopic+study+of+the+human+fallopian+tube.+Report+II.+Fetal+life,+reproductive+life,+and+postmenopause.">Scanning electron microscopic study of the human fallopian tube. Report II. Fetal life, reproductive life, and postmenopause.</a> Fert Steril. 23 (10), 719-733 (1972).</li><li>Coan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+role+of+fallopian+ciliated+cells+in+the+pathogenesis+of+high-grade+serous+ovarian+cancer.">Exploring the role of fallopian ciliated cells in the pathogenesis of high-grade serous ovarian cancer.</a> Int J Mol Sci. 19 (9), 2512 (2018).</li><li>Leese, H., Tay, J., Reischl, J., Downing, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+fallopian+tubal+fluid:+role+of+a+neglected+epithelium.">Formation of fallopian tubal fluid: role of a neglected epithelium.</a> Reproduction. 121 (3), 339-346 (2001).</li><li>Donnez, J., Casanas-Roux, F., Ferin, J., Thomas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+ciliation+and+cell+height+in+human+tubal+epithelium+in+the+fertile+and+post-fertile+years.">Changes in ciliation and cell height in human tubal epithelium in the fertile and post-fertile years.</a> Maturitas. 5 (1), 39-45 (1983).</li><li>Lengyel, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+atlas+of+the+human+postmenopausal+fallopian+tube+and+ovary+from+single-cell+RNA+and+ATAC+sequencing.">A molecular atlas of the human postmenopausal fallopian tube and ovary from single-cell RNA and ATAC sequencing.</a> Cell Rep. 41 (12), 111838-111838 (2022).</li><li>Tao, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+tubal+ciliated+cells+as+a+risk+for+&amp;#34;ovarian&amp;#34;+or+pelvic+serous+carcinoma.">Loss of tubal ciliated cells as a risk for &amp;#34;ovarian&amp;#34; or pelvic serous carcinoma.</a> Am J Cancer Res. 10 (11), 3815 (2020).</li><li>Li, J., Fadare, O., Xiang, L., Kong, B., Zheng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+serous+carcinoma:+recent+concepts+on+its+origin+and+carcinogenesis.">Ovarian serous carcinoma: recent concepts on its origin and carcinogenesis.</a> J Hematol Oncol. 5 (1), 8 (2012).</li><li>Hu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+repertoire+of+serous+ovarian+cancer+non-genetic+heterogeneity+revealed+by+single-cell+sequencing+of+normal+fallopian+tube+epithelial+cells.">The repertoire of serous ovarian cancer non-genetic heterogeneity revealed by single-cell sequencing of normal fallopian tube epithelial cells.</a> Cancer Cell. 37 (2), 226-242.e7 (2020).</li><li>Dinh, H. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptomics+identifies+gene+expression+networks+driving+differentiation+and+tumorigenesis+in+the+human+fallopian+tube.">Single-cell transcriptomics identifies gene expression networks driving differentiation and tumorigenesis in the human fallopian tube.</a> Cell Rep. 35 (2), 108978 (2021).</li><li>Ulrich, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+heterogeneity+of+human+fallopian+tubes+in+normal+and+hydrosalpinx+disease+states+identified+using+scRNA-seq.">Cellular heterogeneity of human fallopian tubes in normal and hydrosalpinx disease states identified using scRNA-seq.</a> Dev Cell. 57 (7), 914-929.e7 (2022).</li><li>Brand, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fallopian+tube+single+cell+analysis+reveals+myeloid+cell+alterations+in+high-grade+serous+ovarian+cancer.">Fallopian tube single cell analysis reveals myeloid cell alterations in high-grade serous ovarian cancer.</a> iScience. 27 (3), 108990-108990 (2024).</li><li>Memarzadeh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-autonomous+activation+of+the+PI3-kinase+pathway+initiates+endometrial+cancer+from+adult+uterine+epithelium.">Cell-autonomous activation of the PI3-kinase pathway initiates endometrial cancer from adult uterine epithelium.</a> Proc Natl Acad Sci USA. 107 (40), 17298-17303 (2010).</li><li>Cunha, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+induction+and+specification+of+morphogenesis+and+cytodifferentiation+of+the+epithelia+of+the+mullerian+ducts+and+urogenital+sinus+during+development+of+the+uterus+and+vagina+in+mice.">Stromal induction and specification of morphogenesis and cytodifferentiation of the epithelia of the mullerian ducts and urogenital sinus during development of the uterus and vagina in mice.</a> J Exp Zool. 196 (3), 361-369 (1976).</li><li>Xie, Y., Park, E. -. S., Xiang, D., Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+organoid+culture+reveals+enrichment+of+organoid-forming+epithelial+cells+in+the+fimbrial+portion+of+mouse+fallopian+tube.">Long-term organoid culture reveals enrichment of organoid-forming epithelial cells in the fimbrial portion of mouse fallopian tube.</a> Stem Cell Res. 32 (1873-5061), 51-60 (2018).</li><li>Karst, A. M., Drapkin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+immortalization+of+human+fallopian+tube+secretory+epithelial+cells.">Primary culture and immortalization of human fallopian tube secretory epithelial cells.</a> Nat Protoc. 7 (9), 1755-1764 (2012).</li><li>Ford, M. J., Harwalkar, K., Yamanaka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+to+generate+mouse+oviduct+epithelial+organoids+for+viral+transduction+and+whole-mount+3D+imaging.">Protocol to generate mouse oviduct epithelial organoids for viral transduction and whole-mount 3D imaging.</a> STAR Protocols. 3 (1), 101164 (2022).</li><li>Radecki, K. C., Lorenson, M. Y., Carter, D. G., Walker, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdissection+and+dissociation+of+the+murine+oviduct:+Individual+segment+identification+and+single+cell+isolation.">Microdissection and dissociation of the murine oviduct: Individual segment identification and single cell isolation.</a> J Vis Exp. (177), e63168 (2021).</li><li>Feng, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+the+detection+of+organoid-initiating+cell+activity+in+patient-derived+single+fallopian+tube+epithelial+cells.">Protocol for the detection of organoid-initiating cell activity in patient-derived single fallopian tube epithelial cells.</a> Methods Mol Bio. 2429, 445-454 (2022).</li><li>Kessler, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Notch+and+Wnt+pathways+regulate+stemness+and+differentiation+in+human+fallopian+tube+organoids.">The Notch and Wnt pathways regulate stemness and differentiation in human fallopian tube organoids.</a> Nat Commun. 6 (1), 8989 (2015).</li><li>Fotheringham, S., Levanon, K., Drapkin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+culture+of+primary+human+fallopian+tube+epithelial+cells.">Ex vivo culture of primary human fallopian tube epithelial cells.</a> J Vis Exp. (51), e2728 (2011).</li></ol>

<table><tbody><tr><td>1% Trypsin&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>J63993.09</td><td></td></tr><tr><td>100 &amp;micro;m Cell Strainer</td><td>Corning</td><td>431752</td><td></td></tr><tr><td>4% Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710-S</td><td></td></tr><tr><td>5 mL round bottom tubes</td><td>Corning</td><td>352008</td><td></td></tr><tr><td>60 mm cell culture plate</td><td>Corning&amp;nbsp;</td><td>CLS430589-500EA</td><td></td></tr><tr><td>6-well Cell Culture Plate</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>Anti-CD10</td><td>BioLegend</td><td>312212</td><td></td></tr><tr><td>Anti-CD326 (Ep-CAM)</td><td>BioLegend</td><td>324218</td><td></td></tr><tr><td>Anti-CD45&amp;nbsp;</td><td>BioLegend</td><td>304026</td><td></td></tr><tr><td>Anti-EpCAM</td><td>Abcam</td><td>ab223582</td><td></td></tr><tr><td>Anti-IgG2a PerCP&amp;nbsp;</td><td>BioLegend</td><td>400250</td><td></td></tr><tr><td>Anti-PAX8</td><td>Sigma-Aldrich</td><td>363M-15</td><td></td></tr><tr><td>Anti-Vimentin</td><td>Agilent Technologies</td><td>M072501-2</td><td></td></tr><tr><td>Chamber Slide System</td><td>Thermo Scientific&amp;nbsp;</td><td>154917PK</td><td></td></tr><tr><td>Collagenase&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>17100017</td><td></td></tr><tr><td>DMEM</td><td>Thermo Scientific&amp;nbsp;</td><td>10569-010</td><td></td></tr><tr><td>DNase I</td><td>Sigma</td><td>10104159001</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>3690</td><td></td></tr><tr><td>eFluor 506</td><td>Invitrogen</td><td>65-0866-14</td><td></td></tr><tr><td>FBS</td><td>Sigma</td><td>F2442</td><td></td></tr><tr><td>Fine point forceps&amp;nbsp;</td><td>VWR</td><td>102091-526</td><td>Any finepoint forceps of your choice will work</td></tr><tr><td>Fixable Viability Dye eFluor 506</td><td>Invitrogen</td><td>65-0866-14</td><td></td></tr><tr><td>FlowJo software version 9&amp;nbsp;</td><td>BD Biosciences</td><td></td><td>Data analysis software</td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>HBSS</td><td>Thermo Scientific&amp;nbsp;</td><td>14175-095</td><td></td></tr><tr><td>MACSQuant Analyzer 10 flow cytometer&amp;nbsp;</td><td>Miltenyi Biotec</td><td></td><td></td></tr><tr><td>MACSQuant Calibration Beads</td><td>Miltenyi Biotec</td><td>130-093-607</td><td></td></tr><tr><td>Mammocult&amp;nbsp;</td><td>Stemcell Technologies</td><td>5620</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Fisher Scientific</td><td>PI31873</td><td></td></tr><tr><td>PBS&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>14190-144</td><td></td></tr><tr><td>Penicillin-Streptomyocin&amp;nbsp;</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>PerCP Conjugated CD45</td><td>BioLegend</td><td>304026</td><td></td></tr><tr><td>Red Blood Cell lysis buffer&amp;nbsp;</td><td>Tonbo Biosciences</td><td>TNB-4300-L100&amp;nbsp;</td><td></td></tr><tr><td>Triton X-100</td><td>Thermo Scientific&amp;nbsp;</td><td>BP151-100</td><td></td></tr><tr><td>Vannas-Tubingen Spring Scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>15003-08</td><td></td></tr><tr><td>VECTASHIELD with DAPI</td><td>Fisher Scientific</td><td>NC9524612</td><td></td></tr></tbody></table>

an efficient method for extracting human fallopian tube epithelia for single-cell analyses

提取人输卵管上皮细胞用于单细胞分析的有效方法

Human fallopian tubes are intrinsic to reproduction. The purpose of the fallopian tubes is to allow for the transit of sperm, the ovum, and if ...

an-efficient-method-for-extracting-human-fallopian-tube-epithelia-for

Research

JoVE Journal

Cancer Research

An Efficient Method for Extracting Human Fallopian Tube Epithelia for Single-cell Analyses

349 次观看.  University of California, Los Angeles. 该方案描述了一种用于分离人输卵管上皮细胞的一天方法。分离的上皮细胞可以接种在 2 维 （2D） 培养物中，也可以解离成单细胞悬液，并用于下游实验，包括流式细胞术和单细胞 RNA 测序。 

视频: 提取人输卵管上皮细胞用于单细胞分析的有效方法

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types.

内髂动脉注射显微骨转移的效率和选择性建模

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

科研

癌症研究

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue organization. Drosophila melanogaster has emerged as a popular experimental model system in cancer biology to study the genetic and cellular mechanisms of tumorigenesis. In particular, genetic tools for Drosophila imaginal discs (developing epithelia in larvae) enable the creation of transformed pro-tumor cells within a normal epithelial tissue, a situation similar to the initial stages of human cancer. A recent study of tumorigenesis in Drosophila wing imaginal discs, however, showed that tumor initiation depends on the tissue-intrinsic cytoarchitecture and the local microenvironment, suggesting that it is important to consider the region-specific susceptibility to tumorigenic stimuli in evaluating tumor phenotypes in imaginal discs. To facilitate phenotypic analysis of tumor progression in imaginal discs, here we describe a protocol for genetic experiments using the GAL4-UAS system to induce neoplastic tumors in wing imaginal discs. We further introduce a diagnosis method to classify the phenotypes of clonal lesions induced in imaginal epithelia, as a clear classification method to discriminate various stages of tumor progression (such as hyperplasia, dysplasia, or neoplasia) had not been described before. These methods might be broadly applicable to the clonal analysis of tumor phenotypes in various organs in Drosophila.

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

Mosaic clone analysis in Drosophila imaginal disc epithelia is a powerful model system to study the genetic and cellular mechanisms of tumorigenesis. Here we describe a protocol to induce tumors in Drosophila wing imaginal discs using the GAL4-UAS system, and introduce a diagnosis method to classify the tumor phenotypes.

诱导和诊断肿瘤<em&gt;果蝇</em&gt; Imaginal Disc Epithelia

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

单细胞下游特征的靶细胞预富集与全基因组扩增

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

二维凝胶电泳结合质谱法分析人垂体腺瘤组织蛋白质组

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

应用 Fraumeni 综合征诱导多潜能干细胞建模骨肉瘤

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and protein functions, and cell signaling aberrations may therefore serve as biomarkers to indicate personalized treatment options. As opposed to DNA and RNA analyses, changes in protein activity can more efficiently evaluate the mechanisms underlying drug sensitivity and resistance. Phospho flow cytometry is a powerful technique that measures protein phosphorylation events at the cellular level, an important feature that distinguishes this method from other antibody-based approaches. The method allows for simultaneous analysis of multiple signaling proteins. In combination with fluorescent cell barcoding, larger medium- to high-throughput data-sets can be acquired by standard cytometer hardware in short time. Phospho flow cytometry has applications both in studies of basic biology and in clinical research, including signaling analysis, biomarker discovery and assessment of pharmacodynamics. Here, a detailed experimental protocol is provided for phospho flow analysis of purified peripheral blood mononuclear cells, using chronic lymphocytic leukemia cells as an example.

Here, a protocol for medium- to high-throughput analysis of protein phosphorylation events at the cellular level is presented. Phospho flow cytometry is a powerful approach to characterize signaling aberrations, identify and validate biomarkers, and assess pharmacodynamics.

磷流式细胞仪与荧光细胞条形码用于单细胞信号分析和生物标志物发现

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and ...

An Automated Microscopic Scoring Method for the &#947;-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects induced by exposure to ionizing radiation to make an individual dose assessment. This is pertinent in the framework of radiation emergencies, where health assessments and planning of clinical treatment for exposed victims are critical. Since DNA double strand breaks (DSB) are considered to be the most lethal form of radiation-induced DNA damage, this protocol presents a method to detect DNA DSB in blood samples. The methodology is based on the detection of a fluorescent labelled phosphorylated DNA repair protein, namely, &#947;-H2AX. The use of an automated microscopy platform to score the number of &#947;-H2AX foci per cell allows a standardized analysis with a significant decrease in the turn-around time. Therefore, the &#947;-H2AX assay has the potential to be one of the fastest and sensitive assays for biological dosimetry. In this protocol, whole blood samples from healthy adult volunteers will be processed and irradiated in vitro in order to illustrate the usage of the automated and sensitive &#947;-H2AX foci assay for biodosimetry applications. An automated slide scanning system and analysis platform with an integrated fluorescence microscope is used, which allows the fast, automatic scoring of DNA DSB with a reduced degree of bias.

This protocol presents the slide preparation and automated scoring of the &#947;-H2AX foci assay on peripheral blood lymphocytes. To illustrate the method and sensitivity of the assay, isolated lymphocytes were irradiated in vitro. This automated method of DNA DSB detection is useful for fast and high-throughput biological dosimetry applications.

一种人外周血淋巴细胞γ-H2AX病灶测定的自动显微镜评分方法

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects ...

Subculture and Cryopreservation of Esophageal Adenocarcinoma Organoids: Pros and Cons for Single Cell Digestion

The lack of suitable translational research models reflecting primary disease to explore tumorigenesis and therapeutic strategies is a major obstacle in esophageal adenocarcinoma (EAC). Patient-derived organoids (PDOs) have recently emerged as a remarkable preclinical model in a variety of cancers. However, there are still limited protocols available for developing EAC PDOs. Once the PDOs are established, the propagation and cryopreservation are essential for further downstream analyses. Here, two different methods have been standardized for EAC PDOs subculture and cryopreservation, i.e., with and without single cell digestion. Both methods can reliably obtain appropriate cell viability and are applicable for a diverse experimental setup. The current study demonstrated that subculturing EAC PDOs with single cell digestion is suitable for most experiments requiring cell number control, uniform density, and a hollow structure that facilitates size tracking. However, the single cell-based method shows slower growth in culture as well as after re-cultivation from frozen stocks. Besides, subculturing with single cell digestion is characterized by forming hollow structures with a hollow core. In contrast, processing EAC PDOs without single cell digestion is favorable for cryopreservation, expansion, and histological characterization. In this protocol, the advantages and disadvantages of subculturing and cryopreservation of EAC PDOs with and without single cell digestion are described to enable researchers to choose an appropriate method to process and investigate their organoids.

This protocol describes the methods of subculture and cryopreservation of esophageal adenocarcinoma organoids with and without single cell digestion to enable researchers to choose appropriate strategies based on their experimental design.

食管腺癌类器官的传代培养和冷冻保存:单细胞消化的利弊

The lack of suitable translational research models reflecting primary disease to explore tumorigenesis and therapeutic strategies is a major obstacle in ...

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

从正常和肿瘤乳腺组织中生成和成像小鼠和人上皮类器官，无需传代

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's interstitial space. The efficient enrichment of these sEVs is an important prerequisite to the study of their biological function and a key to the development of clinical detection techniques and therapeutic carrier technology. It is difficult to isolate sEVs from tissue because they are usually heavily contaminated. This study provides a method for the rapid enrichment of high-quality sEVs from liver cancer tissue. The method involves a four-step process: the incubation of digestive enzymes (collagenase D and DNase &#921;) with tissue, filtration through a 70 &#181;m cell strainer, differential ultracentrifugation, and filtration through a 0.22 &#181;m membrane filter. Owing to the optimization of the differential ultracentrifugation step and the addition of a filtration step, the purity of the sEVs obtained by this method is higher than that achieved by classic differential ultracentrifugation. It provides an important methodology and supporting data for the study of tissue-derived sEVs.

Here we describe the enrichment of small extracellular vesicles derived from liver cancer tissue through an optimized differential ultracentrifugation method.

肝癌组织来源于小细胞外囊泡的富集方法

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's ...