| Isolation and in vivo Transfer of Antigen Presenting Cells
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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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| In vitro Tumorsphere Formation Assays
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Author:
Date:
2013-02-05
[Abstract] A tumorsphere is a solid, spherical formation developed from the proliferation of one cancer stem/progenitor cell. These tumorspheres (Figure 1a) are easily distinguishable from single or aggregated cells (Figure 1b) as the cells appear to become fused together and individual cells cannot be identified. Cells are grown in serum-free, non-adherent conditions in order to enrich the cancer stem/progenitor cell population as only cancer stem/progenitor cells can survive and proliferate in this environment. This assay can be used to estimate the percentage of cancer stem/progenitor cells present in a population of tumor cells. The size, which can vary from less than 50 micrometers to 250 micrometers, and number of tumorspheres formed can be used to characterize the cancer stem/progenitor cell ...
[摘要] 肿瘤球是从一个癌干细胞/祖细胞的增殖形成的固体，球形形式。这些肿瘤球（图1a）易于与单个或聚集的细胞（图1b）区分开，因为细胞看起来融合在一起并且不能鉴定个体细胞。细胞在无血清，非粘附条件下生长以富集癌干细胞/祖细胞群，因为只有癌干/祖细胞可在这种环境中存活和增殖。该测定可用于估计存在于肿瘤细胞群中的癌干细胞/祖细胞的百分比。可以从小于50微米至250微米变化的大小和形成的肿瘤球的数目可以用于表征体外培养的癌细胞群内的癌干/祖细胞群，以及在体内肿瘤（Lo等人，2012; Liu等人，2009）。虽然几种细胞系可用于肿瘤球形成测定（例如来自Her2/neu转基因小鼠，MCF7，BT474和HCC1954的原发性乳腺肿瘤细胞），但一些细胞系可能不形成典型的肿瘤球结构，并且可能难以计数或最终分类为肿瘤球。
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| Sphere Formation (Osteosphere/Sarcopshere) Assay
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Author:
Date:
2012-12-20
[Abstract] Self-renewing cells from adult tissue (such as bone) that represent a progenitor population can be grown in suspension cultures in the presence of defined serum-free medium. Progenitor cells can be identified by this property of anchorage-independent growth in suspension cultures. These spherical clusters of progenitor bone cells growing under non-adherent conditions are called osteospheres. Such progenitor populations often possess characteristics of multipotency and can differentiate into multiple mesenchymal lineages. Cancer cells capable of growing in suspension have also been reported in osteosarcomas, tumors of the bone tissue. These spherical colonies formed from single cells (clonal) in non-adherent conditions are generally considered to represent self-renewing, stem-like cells ...
[摘要] 来自代表祖细胞群体的成体组织（例如骨）的自我更新细胞可以在确定的无血清培养基的存在下在悬浮培养物中生长。 祖细胞可以通过悬浮培养物中锚定非依赖性生长的这种性质来鉴定。 这些在非粘附条件下生长的祖细胞骨细胞的球形簇称为骨干球。 这样的祖细胞群通常具有多能性的特征并且可以分化成多个间充质谱系。 在骨肉瘤，骨组织的肿瘤中也报道了能够在悬浮液中生长的癌细胞。 在非粘附条件下由单细胞（克隆）形成的这些球形菌落通常被认为代表自我更新的干细胞样细胞，并且可以用于其它测定，例如多能性和有限稀释分析（LDA）。
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