Name: preparation of myeloid derived suppressor cells (mdsc) from naive and pancreatic tumor-bearing mice using flow cytometry and automated magnetic activated cell sorting (automacs)
Uploaded: 2012-06-18T13:00:00
Description: Watch this Scientific Journal Video about Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell S

我们承认USF流式细胞仪核心设施。我们想感谢丹尼斯库珀博士资源共享。我们也想感谢他们的协助玛雅科恩，劳拉·彭德尔顿和戴安娜拉图尔建立和拍摄这部影片。神经网络的支持NSF FG-LSAMP桥博士奖学金人力资源开发＃0929435。这项工作是由美国癌症学会院校研究资助＃93-032-13/Moffitt癌症中心颁发到TG。

开始之前，请准备以下解决方案： 3％，染色媒体（SM）： -3％的胎牛血清（FBS），1X的磷酸盐缓冲液（PBS） 磁珠缓存（MB）： - 牛血清白蛋白（BSA）的0.5％白蛋白在1XPBS 1。收获从小鼠脾脏<ol><li>皮下注入1.5×10 5小鼠Panc02细胞悬浮在100μL1X PBS 6-8周岁的C57BL / 6...

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这是一个详细的方法处理和immunophentyping的MDSC人口，是适用于各种动物模型不同的淋巴组织。特别，autoMACS富集，可用于各种白细胞的人口，包括GR-1枯竭脾4，从脾和淋巴结肿大5粒细胞亚群的净化，分 ​​离骨髓中性粒细胞14和纯化的CD8 + T细胞从脾隔离和淋巴结15。无论所需的淋巴组织产生白细胞的单细胞悬液的细胞群体的利益，需要最佳表面染色，流式?...

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 目录号 </td><td> 评论 </td></tr><tr><td> 1X磷酸盐缓冲液</td><td>热科学胎牛血清</td><td> SH30028.02 </td><td>的Ca 2 + /镁2 + /苯酚红</td></tr><tr><td>白蛋白牛血清白蛋白（BSA） </td><td> Sigma-Aldrich公司</td><td> A7906 </td><td>让牛血清白蛋白溶解在PBS原状;无菌环境</td></tr><tr><td>胎牛血清（FBS）， </td><td>热科学胎牛血清</td><td> SV3001403HI </td><td>热灭活;无菌环境</td></tr><tr><td>大鼠抗小鼠单克隆抗体CD16/32（FC块） </td><td> BD公司</td><td> 553142 </td><td>无菌环境</td></tr><tr><td>抗鼠细胞CD11b（MAC-1）FITC标记</td><td> eBiosciences </td><td> 11-0112 </td><td>不育环境</td></tr><tr><td>抗鼠Ly6G（GR-1）APC </td><td> eBiosciences </td><td> 17-5931 </td><td>无菌环境</td></tr><tr><td>抗鼠Ly6G（GR-1）PE </td><td> eBiosciences </td><td> 12-5931 </td><td>无菌环境</td></tr><tr><td>的DAPI </td><td> Invitrogen公司</td><td> D1306 </td><td>连续稀释无菌环境</td></tr><tr><td>细胞分离筛</td><td> Sigma-Aldrich公司</td><td> CD1-1KT </td><td>使用高压灭菌前</td></tr><tr><td> 70微米过滤器</td><td> BD公司</td><td> 352350 </td><td>无菌环境</td></tr><tr><td> 1X红细胞裂解液</td><td> eBiosciences </td><td> 00-4333-57 </td><td>温暖到室温后使用无菌环境</td></tr><tr><td>培养皿</td><td> Fisher Scientific则</td><td> 08-757-12 </td><td>无菌环境</td></tr><tr><td> 50毫升CONICAL管</td><td> Thermo Scientific的</td><td> 339652 </td><td>无菌环境</td></tr><tr><td> 5毫升12X75mm聚苯乙烯圆底管</td><td> BD公司</td><td> 352054 </td><td>无菌环境已知流式细胞仪管; </td></tr><tr><td> 96-V型底板</td><td>康宁</td><td> 3897 </td><td>无菌环境</td></tr><tr><td>台盼蓝</td><td> cellgro </td><td> 25-900-CI </td><td>无菌环境</td></tr><tr><td>体育微珠</td><td>美天旎生物技术</td><td> 130-048-801 </td><td>无菌环境</td></tr><tr><td> AutoMACS临分离</td><td>美天旎生物技术</td><td> 130-092-545 </td><td></td></tr><tr><td> AutoMACS列</td><td>美天旎生物技术</td><td> 130-021-101 </td><td></td></tr><tr><td> AutoMACS运行缓冲液</td><td>美天旎生物技术</td><td> 130-091-221 </td><td&gt; </td></tr></tbody></table>

preparation of myeloid derived suppressor cells (mdsc) from naive and pancreatic tumor-bearing mice using flow cytometry and automated magnetic activated cell sorting (automacs)

的MDSC是一个不成熟的巨噬细胞，树突状细胞和粒细胞积聚在病理条件下的淋巴器官，包括寄生虫感染，炎症，创伤后应激，移植物抗宿主病，糖尿病和癌症1-7异构人口。在小鼠的MDSC明示的Mac-1（细胞CD11b）和GR-1（Ly6G和Ly6C）表面抗原。重要的是要注意的MDSC荷，它们显着扩大在各个主机研究和压制7-10天真对口相比抗肿瘤免疫反应。然而，根据病理状态，有不同的MDSC亚与不同的机制和抑制11,12的目标。因此，有效的方法来隔离可行的MDSC人口是重要的，在阐明各自不同的分子机制抑制在体外和体内 。 近日，在Ghansah组报告的MDSC在小鼠胰腺癌模型的扩展。我们的肿瘤轴承的MDSC显示动态平衡的损失，并增加抑制功能比较幼稚的MDSC 13。的MDSC的百分比显着天真与荷瘤小鼠的淋巴车厢。这是一个重大的警告，这往往阻碍了这些的MDSC准确的比较分析。因此，丰富天真小鼠GR-1 +白细胞荧光激活细胞分选（FACS）前提高纯度，活力和排序的时间显着降低。然而，丰富的GR-1 +荷瘤小鼠的白细胞是可选的，因为这些丰富的排序快速流式细胞仪。因此，在这个协议中，我们描述了一个高效的方法，免疫的MDSC从排序的MDSC及时天真小鼠脾脏和丰富的Gr-1 +白细胞。免疫C57BL / 6小鼠接种小鼠Panc02 CE的LLS皮下而天真的小鼠接受1XPBS。大约30天接种后脾脏被采伐和加工成单细胞悬液采用细胞分离筛。脾然后红细胞（RBC）的裂解，这些白细胞等分对MAC-1和GR-1免疫的MDSC百分比用流式细胞仪使用荧光标记抗体染色。在一个类似的实验，从整个天真的小鼠白细胞染色与荧光标记的GR-1抗体，培养与PE的微珠和正选择使用一个自动化的磁性活化细胞分选（autoMACS）分离。接下来，GR-1 +白细胞等份与Mac-1抗体染色，以确定在使用流式细胞仪的MDSC百分比增加。现在，这些GR1 +丰富的白细胞是准备用于比较分析（天真与荷瘤） 在体内和体外的MDSC排序流式细胞仪</em&gt;的检测。

Watch this Scientific Journal Video about Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell S

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

这是一个迅速和全面的免疫髓源性抑制细胞（MDSC）和丰富GR-1的方法<sup&gt; -</sup&gt;从小鼠脾脏白细胞。这种方法使用流式细胞仪和AutoMACS细胞排序丰富可行的GR-1<sup&gt; -</sup&gt;白细胞之前使用的MDSC排序，以流式细胞仪<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;检测。

制备髓源性抑制细胞从幼稚和胰腺癌荷瘤小鼠，用流式细胞仪和自动磁性细胞分选（AutoMACS）（MDSC）

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

Research

JoVE Journal

Medicine

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

Magnetic Resonance Derived Myocardial Strain Assessment Using Feature Tracking

磁共振衍生的心肌劳损评估使用特征跟踪

Purpose: An accurate and practical method to measure parameters like strain in myocardial tissue is of great clinical value, since it has been shown, that ...

科研

医学

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

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Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

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Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

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Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

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Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

半自动适应循环肿瘤细胞（CTC）分析临床和临床前研究中的应用

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

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纳米抗体和基于抗体的验证<em&gt;在体内</em&gt;肿瘤异种移植NIRF成像的小鼠实验使用<em&gt;离体</em&gt;流式细胞仪和显微镜

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry

分子探针的优化，以确定细胞死亡的光合生物（<em&gt;铜绿微囊藻</em&gt;）使用流式细胞仪

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. ...