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Author:
Date:
2014-10-05
[Abstract] Transfer of antigen presenting cells in vivo is a method used by immunologists to examine the potency of antigen presentation by a selected population of cells. This method is most commonly used to analyze presentation of protein antigens to MHC class I or II restricted T cells, but it can also be used for studies of nonconventional antigens such as CD1-presented lipids. In a recent study focusing on CD1d-restricted glycolipid antigen presentation to Natural Killer T cells, we compared antigen presenting properties of splenic B cells, CD8αPos dendritc cells (DCs) and CD8αNeg DCs (Arora et al., 2014). This protocol describes the detailed method used for isolation of these cell populations, and their transfer into recipient mice to analyze their ...
[摘要] 体内转移抗原呈递细胞是免疫学家用来检查所选择的细胞群体的抗原呈递效力的方法。该方法最常用于分析蛋白质抗原对MHC I类或II类限制性T细胞的呈递，但其也可用于非常规抗原如CD1呈递脂质的研究。在最近关于CD1d限制性糖脂抗原呈递到天然杀伤T细胞的研究中，我们比较脾B细胞，CD8αpos 树突细胞（DC）和CD8α Neg sup> DC（Arora et al。，2014）。该方案描述了用于分离这些细胞群体和将其转移到受体小鼠中以分析其抗原呈递性质的详细方法。作为总单核细胞的百分比，平均脾脏含有约1-3％骨髓树突状细胞（DC）。在绝对数量上，这转换为大约0.6-1.8×10 6个DC。为了增强脾中DC的数目，向小鼠皮下注射来自经过工程改造以表达fms相关的酪氨酸激酶3配体（Flt3L）的培养的黑素瘤细胞系（B16.Flt3L）的细胞（Mach等人。，2000）。该蛋白是与集落刺激因子-1同源的生长因子，并在造血干细胞的分化中起关键作用。将这种蛋白质作为纯化的蛋白质施用到小鼠中导致多个器官中的CD8αpos pos和CD8α阳性DC亚群的扩增。在已经植入过表达这种蛋白质的肿瘤细胞的小鼠中也观察到类似的扩增（Mach等人，2000）。根据我们的经验，来自具有可触知的B16.Flt3L肿瘤的小鼠的脾脏中高达60％的总单核细胞可以是CD11c阳性树突细胞，从而得到高达5×10 ...
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Author:
Date:
2014-09-20
[Abstract] Immune cells, such as microglia are resident in the brain and spinal cord of normal mice and humans. Furthermore, macrophages, dendritic cells, T cells, B cells and NK cells infiltrate the CNS during certain infections or in neurodegenerative/neuroinflammatory diseases, such as experimental autoimmune encephalomyelitis (EAE) (a model for multiple sclerosis) or Alzheimer’s disease (Sutton et al., 2009; Browne et al., 2013). Infiltrating cells can be identified using immunohistological staining of sections from brain or spinal cords. However, more detailed phenotypic and functional analysis is possible following isolation of the immune cells from the CNS of normal or diseased mice. Purification of mononuclear cells from brain or spinal cord is dependent on perfusing the ...
[摘要] 免疫细胞，例如小神经胶质细胞存在于正常小鼠和人的脑和脊髓中。此外，巨噬细胞，树突细胞，T细胞，B细胞和NK细胞在某些感染或神经变性/神经炎症疾病如实验性自身免疫性脑脊髓炎（EAE）（多发性硬化症模型）或阿尔茨海默病（Sutton >等人，2009; Browne等人，2013）。可以使用来自脑或脊髓的切片的免疫组织学染色来鉴定浸润细胞。然而，在从正常或患病小鼠的CNS中分离免疫细胞后，可能进行更详细的表型和功能分析。来自脑或脊髓的单核细胞的纯化取决于灌注小鼠以确保在离解组织和在Percoll梯度上纯化单核细胞之前从CNS组织中除去血液。该技术提供了具有高存活力的细胞的单细胞悬浮液，其适合于FACS分析或有限的功能研究。产率通常低于正常小鼠脑或脊髓，但从具有EAE或CNS感染的小鼠的产量更高。当与细胞内细胞因子染色和FACS组合时，该技术特别用于分析致病性T细胞（Th17和Th1细胞）及其在EAE中的调节/调节。
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