| Quantification of Tumor Material Uptake
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Author:
Date:
2016-10-20
[Abstract] Extracellular tumor material including exosomes, microvesicles and apoptotic tumor debris may help cancers invade new organs. Enhancing the removal of extracellular tumor material by immune cells represents a novel immunotherapy approach for preventing cancer metastasis. This protocol quantifies the uptake and removal of extracellular tumor material from circulation and tissues by immune cells. In this assay fluorescent tumor cells are transferred into mice, and then immune cells are quantified by either flow cytometry or imaging cytometry for their uptake of tumor material.
[摘要] 包括外来体，微泡和凋亡性肿瘤碎片的细胞外肿瘤材料可以帮助癌症侵入新器官。增强免疫细胞对细胞外肿瘤材料的去除代表了用于预防癌症转移的新型免疫治疗方法。该方案定量免疫细胞从循环和组织吸收和去除细胞外肿瘤物质。在该测定中，将荧光肿瘤细胞转移到小鼠中，然后通过流式细胞术或成像细胞计量术来定量免疫细胞对肿瘤材料的摄取。
[背景] 研究已经证明，包括从肿瘤脱落的外来体，微泡和凋亡性肿瘤碎片的细胞外肿瘤材料是肿瘤转移，生长和逃避免疫应答的重要介质（Vader等人，2014; Pucci和Pittet ，2013; Pucci等人，2016）。免疫细胞具有去除，应答和转运这种循环肿瘤材料的能力（Hanna等人，2015; Pucci等人，2016; Headley等人。，2016）。该协议提供了一种新的方法来量化特定免疫细胞群体的肿瘤材料摄取，并且可以适于测试调节肿瘤材料摄取的免疫靶标。该协议可以帮助更好地理解对细胞外肿瘤材料的免疫应答，希望最终开发针对癌症发展中的细胞外肿瘤材料的新疗法。
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| Protocol-In vitro T Cell Proliferation and Treg Suppression Assay with Celltrace Violet
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Author:
Date:
2016-01-05
[Abstract] Measurement of the incorporation of radionuclides such as 3H-thymidine is a classical immunological technique for assaying T cell proliferation. However, such an approach has drawbacks beyond the inconvenience of working with radioactive materials, such as the inability of bulk radionuclide incorporation measurements to accurately quantitate T cell divisions, and an inability to combine proliferation analyses with simultaneous evaluation of the expression of cellular markers in divided cells. By labeling T cells with reactive dyes such as CFSE, Celltrace Violet, and others that are partitioned equally between daughter cells at each cell division, one can relatively easily track generations of proliferated cells and their expression of various molecules by flow cytometry.
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[摘要] 放射性核素如3 H胸腺嘧啶核苷的掺入的测量是用于测定T细胞增殖的经典免疫学技术。然而，这种方法具有超出使用放射性材料的不便之处的缺点，例如体放射性核素结合测量不能准确地定量T细胞分裂，以及不能结合增殖分析与同时评价分裂的细胞标记物的表达细胞。通过用诸如CFSE，Celltrace Violet等活性染料标记T细胞，在每个细胞分裂的子细胞之间平均分配T细胞，可以通过流式细胞术相对容易地追踪增殖细胞的代和它们的各种分子的表达。 > FoxP3 + 调节性T细胞（Treg）是免疫耐受的关键介质，其功能评价是表征许多免疫模型的重要步骤（Rudensky，2011）。基于其表面表达的CD25（Treg：CD4 + CD25 + sup/+），已经分离了经典的CD4 + Treg和常规或"应答者"T细胞， ，Tresp：CD4 + CD25 - sup/- ）。然而，我们和其他人已经注意到，CD4 + CD25 - 细胞群表达FoxP3转录因子并具有抑制功能。因此，我们利用转基因FoxP3-EGFP小鼠促进基于EGFP（并因此FoxP3）表达的抑制基因和应答者群体的活性纯化。在这里我们提出我们适应的协议，用于测定调节性T细胞抑制Celltrace紫罗兰标记响应T细胞。
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| FACS-based Satellite Cell Isolation From Mouse Hind Limb Muscles
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Author:
Date:
2015-08-20
[Abstract] Fluorescence Activated Cell Sorting (FACS) is a sensitive and accurate method for purifying satellite cells, or muscle stem cells, from adult mouse skeletal muscle (Liu et al., 2013; Sacco et al., 2008; Tierney et al., 2014). Mechanical and enzymatic digestion of hind limb muscles releases mononuclear muscle cells into suspension. This protocol employs fractionation strategies to deplete cells expressing the cell surface markers CD45, CD31, CD11b and Ly-6A/E-Sca1, both by magnetic separation and FACS-based exclusion, and positively select for cells expressing a7-integrin and CD34. This enables the researcher to successfully enrich satellite cells that uniformly express the paired-box transcription factor Pax7 and are capable of long-term self-renewal, skeletal ...
[摘要] 荧光活化细胞分选（FACS）是用于从成年小鼠骨骼肌纯化卫星细胞或肌肉干细胞的灵敏和精确的方法（Liu等人，2013; Sacco等人， ，2008; Tierney ，，2014）。 后肢肌肉的机械和酶消化将单核肌细胞释放到悬浮液中。 该方案采用分级分离策略，通过磁性分离和基于FACS的排除，耗尽表达细胞表面标记CD45，CD31，CD11b和Ly-6A/E-Sca1的细胞，并阳性选择表达α7-整联蛋白和CD34的细胞。 这使得研究者能够成功地富集均匀表达配对盒转录因子Pax7并且能够长期自我更新，骨骼肌修复和肌肉干细胞池再增殖的卫星细胞。
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