| 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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| Primary Culture of SVZ-derived Progenitors Grown as Neurospheres
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Author:
Date:
2013-08-20
[Abstract] SVZ-derived progenitors grown as neurospheres is a well-known model to study neural stem cell and progenitor functions such as proliferation, differentiation/self-renewal, and migration (Durbec and Rougon, 2001). This protocol is for preparing a culture of SVZ-derived progenitors from 8 early postnatal mouse brains (P0 to P3). One week after cell plating, we can observe round floating neurospheres, each resulting from the clonal expansion of a single EGF/FGF responsive neural progenitor.
[摘要] 作为神经球生长的SVZ衍生的祖细胞是研究神经干细胞和祖细胞功能如增殖，分化/自我更新和迁移的众所周知的模型（Durbec和Rougon，2001）。 该方案用于从8只早期出生后的小鼠脑（P0至P3）制备SVZ衍生祖细胞的培养物。 细胞电镀后一周，我们可以观察到圆形浮游神经球，每个由单个EGF / FGF反应性神经祖细胞的克隆扩增产生。
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