Name: an immunofluorescent method for characterization of barrett’s esophagus cells
Uploaded: 2014-07-20T15:00:00.000+00:00
Duration: 8 min 54 s
Description: Watch this Scientific Journal Video about An Immunofluorescent Method for Characterization of BarrettÃ¢â‚¬â„¢s Esophagus Cells at JoVE.com

这项工作是从圣，若瑟基金会（AJF，LJI）和美国肺脏协会，RG-224607-N（LJI）资助。

1。细胞株维护<ol><li>永生化人是高度不典型增生（CP-B，CP-C，CP-D）和化生（CP-）细胞株的端粒酶稳定转染逆转录酶（TERT）蛋白11。 </li><li>维持在补充有牛垂体提取物（BPE），表皮生长因子（EGF），5％胎牛血清（FBS），非必需氨基酸（NEAA），青霉素 - 链霉素 - 新霉素（PSN）角质形成细胞介质的细胞系，以及标准组织培养条件下（37℃，5％CO 2，98％湿度）。 </li></ol> 2，BE细胞电镀<ol><li>制备12孔板/盖玻片。放置18毫米盖玻片到玻璃培养皿中并高压灭菌。转让蒸压18毫米盖玻片成使用连接到真空管中的无菌玻璃吸管12孔组织培养皿中。用无菌PBS和媒体冲洗盖玻片。 </li><li>胰蛋白酶消化并计数BE永生细胞。计数细胞USI纳克自动细胞计数器或血球。稀释细胞培养基至20,000-40,000个细胞/ ml的浓度。 </li><li>取1 ml萃取池在每孔中含有盖玻片为20,000-40,000个细胞/孔的细胞总数。 </li><li>使用无菌移液管尖端到盖玻片轻轻推到井的底部，以确保细胞附着在盖玻片上。 </li></ol> 3，新药必须经过细胞治疗<ol><li>稀释所选药物在温暖（37℃）介质到所需的浓度和通过反复颠倒试管或用涡旋混合器充分混合。确定药物浓度经验。以下的BE细胞的初始接种，以允许细胞附着在盖玻片开始治疗24小时</li><li>为了便于比较和对照，治疗BE细胞与0.1％的车辆（二甲基亚砜[DMSO]或代理人用于溶解化合物）稀释培养基附加盖玻片。标牌与实验室用笔来识别药品和VEhicle处理的样品。放置细胞到细胞培养孵化器设置在37℃，5％CO 2，98％湿度下孵育期望的时间点。 </li></ol> 4，免疫荧光染色<ol><li>固定术<ol><li>下列药物的治疗所需的温育时间，吸出介质并在室温（RT，25℃）用1×磷酸盐缓冲盐水（PBS）快速洗涤盖玻片。 </li><li>吸出PBS并加入1ml的PBS含4％PFA [4％PFA / PBS]向每个孔中，并在室温下孵育30分钟。 </li><li>吸出4％PFA / PBS中，并用PBS在室温下洗涤​​盖玻片3次，每次5分钟。店铺盖玻片在PBS/0.1％PFA中于4℃，如果需要一个星期。 </li></ol></li><li>透/封锁 通透固定PFA细胞，使抗体来访问胞内靶标。对于细胞外蛋白质浮出水面，执行所有后续步骤而无需皂甙添加到1X PBS/0.5％BSA溶液。 <ol><li>减少背景通过用50mM NH 4 Cl 2稀释在PBS中在RT下10分钟温育和利用RT PBS洗涤一次5分钟。 </li><li>在100-300微升PBS，含0.1％皂角苷和0.5％牛血清白蛋白（BSA）孵育BE细胞30分钟。从过滤灭菌的10％储备稀皂甙，在水中制备并保存于4℃。 </li></ol>注意:这可以防止非特异性的抗体对细胞内蛋白的结合通过阻止由于加入了0.5％BSA的盖玻片。可替换地，代替牛血清白蛋白稀释至5％，以帮助降低抗体的非特异性结合的山羊血清。 </li><li>人力商会的制备<ol><li>通过层叠用湿纸巾的腔室的底部准备一个正方形的生物测定皿，然后将封口膜之上的层。 </li><li>轻轻地用钳子和一个注射器针头朝上盖玻片，细胞，转移到的封口膜。 </li><li>标记的封口膜与实验室笔识别盖玻片。 </li></ol></li><li>初级抗体孵育<ol><li>稀释的一抗在PBS中含有0.5％BSA和0.1％皂苷。 </li><li>用实验室组织轻轻吸去封闭液，加入​​100微升的主要抗体溶液。孵育一抗上的BE细胞过夜，在4℃下</li></ol></li><li>二抗孵育<ol><li>洗了3倍的幻灯片用PBS/0.5％BSA/0.1％皂素为每5分钟在室温。 </li><li>稀释缀合有荧光标记在PBS/0.5％BSA/0.1％皂苷，二次抗体识别，其中第一抗体升高的物种的二级抗体。 </li><li>加入100μl稀释抗体每个盖玻片。孵育二次抗体为2小时，在室温。 </li></ol></li><li>洗涤和盖玻片的安装<ol><li>在PBS/0.5％BSA/0.1％皂素的洗涤盖玻片3次，每次5分钟。除去洗涤缓冲液，并加入100-300微升PBS的盖玻片。 注:对于双反体染色，重复根据在讨论中所述的信息的步骤4.4-4.5与所需的额外初级和次级抗体。 </li><li>使用镊子，轻轻抬起盖玻片，吸去PBS的使用实验室组织或纸巾。 </li><li>加入15微升含4的合适的抗褪色封固剂&#39;，6 - 二脒基-2 - 苯基吲哚（DAPI）的盖玻片和反转盖玻片，将细胞下来，到玻璃显微镜载片上。 </li><li>将幻灯片放入幻灯片的文件夹，并培育远离直射光隔夜在室温，使硬盘设置防褪色媒体治愈，根据制造商的说明。一旦防褪色介质已经设置，幻灯片可以存储在任一4℃或-20℃直到显微可视化。 </li></ol></li></ol>盖玻片5。微观可视化染色的细胞可以通过两种共聚焦显微镜或成像如能直立microscopË。虽然激光共聚焦显微镜提供高清晰度图像离散深处，一个堂堂正正的显微镜能够产生有用的图像的速度更快。为了简便起见，成像的方法染色用标准显微镜呈现BE细胞。在一般情况下，对图像异硫氰酸荧光素（FITC）或类似荧光基团（ 即绿色荧光蛋白），德克萨斯红和DAPI，需要有能够识别这些荧光团的过滤器的显微镜。开始协议来确定兼容的荧光团前​​请检查显微镜。 <ol><li>成像上一个标准的IF显微镜<ol><li>从-20℃或4℃保存删除幻灯片/盖玻片，让他们温至室温。上电镜和汞弧灯。 </li><li>将载玻片与盖玻片朝向目标到显微镜的阶段。使用油浸物镜，加一滴浸油的盖玻片上。 </li><li>选择DAPI过滤器，然后打开快门。 </li&gt; <li>专注于目标，同时通过目镜找，直到图像的显示。由于所有细胞将用DAPI染色，细胞核应便于观察。 </li><li>使用相应的图像处理软件采集图像。 </li></ol></li></ol>

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作者宣称，他们有没有竞争的财务权益。

我们提出的申请，如果来的细胞对阐明在这些细胞的靶向治疗药物的生理效应的方法。尽管我们已经描述了使用对BE细胞这些过程，我们已经发现，这些方法也适用于各种不同类型的细胞13,17,18的。此外，这些程序可以改变多种方式来优化染色的具体目标。 我们发现，在具有适当的中频，直接影响一个参数是固定的选择。我们已经描述了使用4％PFA / PBS中固定在上面的?...

巴雷特食管（BE）是在食管的正常鳞状上皮和慢性暴露于胃食管返流疾病（GERD）1得到的胃内容物的结果化生变化。 BE被认为是针对胃食管反流病的一种保护机制，不过需要的存在赋予食管腺癌（EAC），这种疾病它带有一个显著较差生存1的风险增加。目前的估计表明，美国人口最多5.6％的BE，但是因为BE是常无症状，它被认为是被大多数人仍然未确诊2。由于两个GERD和EAC的发病率已经看到了持续增长3，它已成为重要的是了解参与是EAC的发展的分子机制，特别是因为这些信息可能会提供对预防是选管会的进展的治疗方法。 1的当前模式。慢性炎症，损伤，以及长期暴露于胃内容物造成基因毒性损伤施加的BE病灶促进肿瘤进展到选管会选择压力。许多遗传​​改变已经确定过程中，以选管会的进展。然而，尽管这有一个明显的缺乏了解的细胞信号转导的确切变化，在细胞结构和功能后续影响。 永生体外细胞系是研究细胞信号传导的影响，特别是在调查针对性治疗化合物的作用的有用工具。 BE永生化细胞系的近期商业可用性允许这种研究。虽然高通量测定法，如广泛使用的活力检测，可以在评估靶向疗法后的细胞增殖和苏效果是有价值的rvival 4-6，这些测定是不用于询问细胞信号后的细胞形态的影响是有用的。免疫荧光染色（IF）是用于研究的靶向治疗后细胞的形态学，生长和存活的特性和所涉及的蛋白质的效果的有效技术。我们实验室已经适应了这些方法对永生化BE细胞系，用IF来评价在划定一个可能的化学预防治疗和生物标志物的药物治疗7的希望时BE细胞系在临床可用的药物的作用。同样，在选管会应用这些技术，BE和食管永生化细胞系已划定有关BE选管会的进展8-10关键的发现。我们发现如果用靶向疗法治疗BE细胞的分析具有价值，允许改变细胞结构和蛋白定位的表征。在这里，我们提出我们的方法，如果永生BE细胞的characterize药物治疗。

<table><tbody><tr><td>CP-A (Metaplastic Cell Line)</td><td>ATCC</td><td>CRL-4027</td><td></td></tr><tr><td>CP-B (High-grade Dysplastic Cell Line)</td><td>ATCC</td><td>CRL-4028</td><td></td></tr><tr><td>CP-C (High-grade Dysplastic Cell Line)</td><td>ATCC</td><td>CRL-4029</td><td></td></tr><tr><td>CP-D (High-grade Dysplastic Cell Line)</td><td>ATCC</td><td>CRL-4030</td><td></td></tr><tr><td>Keratinocyte-SFM (1x), Liquid</td><td>Life Technologies</td><td>17005-042</td><td></td></tr><tr><td>0.25% Trypsin-EDTA (1x), Phenol Red&amp;nbsp;</td><td>Life Technologies</td><td>25200056</td><td></td></tr><tr><td>Fetal Bovine Serum, Qualified, HI</td><td>Life Technologies</td><td>10438026</td><td></td></tr><tr><td>PSN antibiotic mixture</td><td>Life Technologies</td><td>15640</td><td></td></tr><tr><td>PBS - Phosphate-Buffered Saline</td><td>Life Technologies</td><td>10010049</td><td></td></tr><tr><td>Pro-long Gold Antifade Reagent with DAPI&amp;nbsp;</td><td>Life Technologies</td><td>P36931</td><td></td></tr><tr><td>Circular Glass Coverslip 18 mm</td><td>Fisher Scientific</td><td>15-183-86</td><td></td></tr><tr><td>&amp;beta;-catenin Primary Antibody (Rabbit)</td><td>Cell Signaling</td><td>9562S</td><td></td></tr><tr><td>Alexa Fluor 488 (Goat Anti-Rabbit)</td><td>Life Technologies</td><td>A11008</td><td></td></tr><tr><td>10 cm TC treated PS dish, sterile</td><td>USA Scientific</td><td>CC7682-3394</td><td></td></tr><tr><td>12-well TC treated PS plate, sterile</td><td>USA Scientific</td><td>5666-5180</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>276855</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma-Aldrich</td><td>A9434</td><td></td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>Bovine Serum Albumin - Fraction V</td><td>Sigma-Aldrich</td><td>85040C</td><td></td></tr><tr><td>SKI-606 (Bosutinib)</td><td>Selleck Chemicals</td><td>S1014</td><td></td></tr><tr><td>Square Bioassay Dish&amp;nbsp;</td><td>Thermo Scientific</td><td>240835</td><td></td></tr><tr><td>Parafilm&amp;nbsp;</td><td>VWR</td><td>82024-546</td><td></td></tr><tr><td>Disposable Pasteur Pipettes, Flint Glass</td><td>VWR</td><td>14672-380</td><td></td></tr><tr><td>Nexcelom Mini Cell Counter</td><td>Nexcelom</td><td></td><td></td></tr><tr><td>Cellometer Counting Chambers</td><td>Nexcelom</td><td>CHT4-SD100-014</td><td></td></tr><tr><td>Zeiss Apotome microscope&amp;nbsp;</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

an immunofluorescent method for characterization of barrett&#8217;s esophagus cells

食管腺癌（EAC）小于17％，总生存率和EAC的发病率急剧上升，在过去的二十年。其中选管会的主要危险因素是Barrett食管（BE），正常食管鳞状在应对慢性胃灼热化生变化。尽管东非共同体之间的行之有效的连接进行的分子事件，涉及的进展特别是改变信号通路的审讯是选管会，了解甚少。这在很大程度上是由于可用来研究这些疾病的体外模型缺乏合适的。最近，永生BE细胞系已成为市售允许在体外研究中BE的。在这里，我们提出了BE永生细胞株的免疫荧光染色的方法，使在细胞信号传导和结构暴露于治疗化合物后体外表征。这些技术的应用将HELp开发洞察参与是EAC的发展机制，并为治疗和预防EAC的潜在途径。

从应用程序的描述的方法获得的结果的一个例子示于图1A-D中 。盖玻片带CP-D和CP-C BE细胞24小时，分别用1微米的Src家族抑制剂，SKI-606，或车辆（DMSO），并使用了粘着连接和Wnt上述程序信号蛋白，β染色-catenin的。 β-连环蛋白的可视化是通过用抗兔IgG结合到绿色荧光标记来实现，而在细胞核的标记是完成了用DAPI（蓝色）标记。盖玻片封片使用硬设置安装并使用激光扫描共聚焦显微镜使?...

Watch this Scientific Journal Video about An Immunofluorescent Method for Characterization of BarrettÃ¢â‚¬â„¢s Esophagus Cells at JoVE.com

An Immunofluorescent Method for Characterization of Barrett&#8217;s Esophagus Cells

有一个明显的需要对Barrett食管的分子驱动程序改进的信息。免疫荧光染色对于理解细胞信号转导的对细胞形态的影响的有效技术。我们提出了一个简单，有效的协议，使用免疫荧光染色，以评估在Barrett食管细胞治疗治疗。

免疫荧光法Barrett食管细胞的表征

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One ...

an-immunofluorescent-method-for-characterization-barretts-esophagus

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.0K 次观看.  St. Joseph's Hospital and Medical Center. 有一个明显的需要对Barrett食管的分子驱动程序改进的信息。免疫荧光染色对于理解细胞信号转导的对细胞形态的影响的有效技术。我们提出了一个简单，有效的协议，使用免疫荧光染色，以评估在Barrett食管细胞治疗治疗。 

视频: An Immunofluorescent Method for Characterization of Barrett’s Esophagus Cells

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

档案福尔马林固定，石蜡包埋的人类口咽鳞状细胞癌样品中T细胞亚群的四色荧光免疫组织化学

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

科研

癌症研究

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification1.
A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs).
The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types2,3. However, no single &#x2018;marker&#x2019; has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. 
A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. 
In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response5, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody6. 
For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells7. The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.

We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray). Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes.

大肠癌细胞表面蛋白的分析，使用一种抗体的芯片和荧光多路复用

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

免疫与感染

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4&#8242;,6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.

A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology studies.

福尔马林固定癌组织标本免疫肿瘤学自动复合免疫荧光小组研究

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of ...

Diagnosis of Neoplasia in Barrett&#8217;s Esophagus using Vital-dye Enhanced Fluorescence Imaging

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat and indistinguishable with white light imaging alone. As a result, a modality that highlights glandular architecture would be useful to discriminate neoplasia from benign epithelium in the distal esophagus. VFI is a novel technique that uses an exogenous topical fluorescent contrast agent to delineate high grade dysplasia and cancer from benign epithelium. Specifically, the fluorescent images provide spatial resolution of 50 to 100 &#956;m&#160;and a field of view up to 2.5 cm,&#160;allowing endoscopists to visualize glandular morphology. Upon excitation, classic Barrett&#8217;s metaplasia appears as continuous, evenly-spaced glands and an overall homogenous morphology; in contrast, neoplastic tissue appears crowded with complete obliteration of the glandular framework. Here we provide an overview of the instrumentation and enumerate the protocol of this new technique. While VFI affords a gastroenterologist with the glandular architecture of suspicious tissue, cellular dysplasia cannot be resolved with this modality. As such, one cannot morphologically distinguish Barrett&#8217;s metaplasia from BE with Low-Grade Dysplasia via this imaging modality. By trading off a decrease in resolution with a greater field of view, this imaging system can be used at the very least as a red-flag imaging device to target and biopsy suspicious lesions; yet, if the accuracy measures are promising, VFI may become the standard imaging technique for the diagnosis of neoplasia (defined as either high grade dysplasia or cancer) in the distal esophagus.

Vital-dye enhanced fluorescence imaging (VFI) is a novel in vivo technique that combines high-resolution epithelial imaging with exogenous topical fluorescent contrast to highlight glandular morphology and delineate neoplasia (high grade dysplasia and cancer) in the distal esophagus.

用生命染增强荧光成像瘤在Barrett食管诊断

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat ...

生物工程

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded&#160;tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image&#160;analysis technique.

多重条形码图像分析，用于石蜡组织样品单细胞分析中的免疫分析和空间映射表征

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

人食管类器官和气液界面培养方案

Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and microbes. Maintaining the integrity of the epithelial barrier is critical for preventing infections and avoiding inflammation caused by harmless food-derived antigens. This article provides simplified protocols for generating human esophageal organoids and air-liquid interface cultures from patient biopsies to study the epithelial compartment of the esophagus in the context of tissue homeostasis and disease. These protocols have been significant scientific milestones in the last decade, describing three-dimensional organ-like structures from patient-derived primary cells, organoids, and air-liquid interface cultures. They offer the possibility to investigate the function of specific cytokines, growth factors, and signaling pathways in the esophageal epithelium within a three-dimensional framework while maintaining the phenotypic and genetic properties of the donor. Organoids provide information on tissue microarchitecture by assessing the transcriptome and proteome after cytokine stimulation. In contrast, air-liquid interface cultures allow the assessment of the epithelial barrier integrity through transepithelial resistance (TEER) or macromolecule flux measurements. Combining these organoids and air-liquid interface cultures is a powerful tool to advance research in impaired esophageal epithelial barrier conditions.

作者聚焦:研究嗜酸性粒细胞性食管炎的病理生理学

Here, a protocol for the culture of human esophageal organoids and air-liquid interface culture is provided. Esophageal organoids' air-liquid interface culture can be used to study the impact of cytokines on the esophageal epithelial barrier.

三维细胞培养模型研究嗜酸性粒细胞性食管炎中上皮屏障 

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

从小鼠和人类食管隔离肌纤维母细胞

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

生物学

Functional Characterization and Visualization of Esophageal Fibroblasts Using Organoid Co-Cultures

Epithelial stem and progenitor cells contribute to the formation and maintenance of the epithelial barrier throughout life. Most stem and progenitor cell populations are tucked away in anatomically distinct locations, enabling exclusive interactions with niche signals that maintain stemness. While the development of epithelial organoid cultures provides a powerful tool for understanding the role of stem and progenitor cells in homeostasis and disease, the interaction within the niche environment is largely absent, thereby hindering the identification of factors influencing stem cell behavior. Fibroblasts play a key role in directing epithelial stem and progenitor fate. Here, a comprehensive organoid-fibroblast co-culture protocol enabling the delineation of fibroblast subpopulations in esophageal progenitor cell renewal and differentiation is presented. In this protocol, a method to isolate both epithelial cells and fibroblasts in parallel from the esophagus is described. Distinct fluorescence-activated cell sorting strategies to isolate both the esophageal progenitor cells as well as the fibroblast subpopulations from either transgenic reporter or wild-type mice are outlined. This protocol provides a versatile approach that can be adapted to accommodate the isolation of specific fibroblast subpopulations. Establishing and passaging esophageal epithelial organoid mono-cultures is included in this protocol, enabling a direct comparison with the co-culture system. In addition, a 3D clearing approach allowing for detailed image analysis of epithelial-fibroblast interactions is described. Collectively, this protocol describes a comparative and relatively high-throughput method for identifying and understanding esophageal stem cell niche components in vitro.

Organoid-fibroblast co-cultures provide a model to study the in vivo stem cell niche. Here, a protocol for esophageal organoid-fibroblast co-cultures is described. Additionally, whole mount imaging is used to visualize the fibroblast-organoid interaction.

使用类器官共培养物对食管成纤维细胞进行功能表征和可视化

Epithelial stem and progenitor cells contribute to the formation and maintenance of the epithelial barrier throughout life. Most stem and progenitor cell ...

免疫荧光法Barrett食管细胞的表征

摘要

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此视频中的章节

档案福尔马林固定，石蜡包埋的人类口咽鳞状细胞癌样品中T细胞亚群的四色荧光免疫组织化学

大肠癌细胞表面蛋白的分析，使用一种抗体的芯片和荧光多路复用

福尔马林固定癌组织标本免疫肿瘤学自动复合免疫荧光小组研究

用生命染增强荧光成像瘤在Barrett食管诊断

多重条形码图像分析，用于石蜡组织样品单细胞分析中的免疫分析和空间映射表征

三维细胞培养模型研究嗜酸性粒细胞性食管炎中上皮屏障

三维细胞培养模型研究嗜酸性粒细胞性食管炎中上皮屏障

三维细胞培养模型研究嗜酸性粒细胞性食管炎中上皮屏障

从小鼠和人类食管隔离肌纤维母细胞

使用类器官共培养物对食管成纤维细胞进行功能表征和可视化