Cell-based Assays to Monitor AID Activity
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Author:
Date:
2016-02-05
[Abstract] The enzyme Activation induced deaminase (AID) underpins antibody affinity maturation and isotype switching through its mutagenic activity of deaminating deoxycytidine to deoxyuridine in DNA. Subsequent processing of the deoxyuridine initiates the processes of somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. Structure-function analysis of AID requires sensitive and biologically relevant methods to measure its various activities. Here we describe simple but effective methods to measure 1) the ability of AID to mutate the Escherichia coli genome, which provides an indication of its catalytic activity; 2) the capacity of AID to perform SHM by complementing a derivative of the DT40 chicken B cell line; 3) the ability of AID to perform CSR by complementing ...
[摘要] 酶活化诱导的脱氨酶(AID)通过其将脱氧胞苷脱氨基到DNA中的脱氧尿苷的诱变活性来支持抗体亲和力成熟和同种型转换。脱氧尿苷的后续加工引发B细胞中体细胞超突变(SHM)和类型转换重组(CSR)的过程。 AID的结构功能分析需要灵敏和生物相关的方法来测量其各种活动。在这里我们描述简单但有效的方法来测量1)AID突变大肠杆菌基因组的能力,其提供其催化活性的指示; 2)AID通过补充DT40鸡B细胞系的衍生物来进行SHM的能力; 3)AID通过补充AID缺乏的原代小鼠B细胞来进行CSR的能力。三种方法的组合,伴随着AID亚细胞定位和蛋白质表达水平和稳定性的必要分析作为对照,允许AID的详细结构功能研究。
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Separation of Intracellular Vesicles for Immunoassays
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Author:
Date:
2015-08-20
[Abstract] The endosome/lysosome systems play important roles in immune cell functions as signaling platforms. Immune cells utilize these endosome/lysosome for signal transduction or intercellular communication to elicit the proper immune responses, regulating the localization or the association of the signaling complexes. Here we introduce the procedures to separate the intracellular vesicles such as endosomes or lysosomes, which could be useful to identify the subcellular localization of the signaling complexes.
[摘要] 内体/溶酶体系统在作为信号平台的免疫细胞功能中起重要作用。 免疫细胞利用这些内体/溶酶体进行信号转导或细胞间通讯,以引发适当的免疫应答,调节信号复合物的定位或缔合。 在这里我们介绍分离细胞内囊泡如内体或溶酶体的程序,这可能有助于识别信号复合物的亚细胞定位。
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Cell Isolation of Spleen Mononuclear Cells
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Author:
Date:
2013-05-05
[Abstract] This method allows you to isolate different subclass mononuclear cells, like B-cells, T cells, CD4+ and CD8+ T, from mouse spleen. By conjugating cells with specific antibodies and subsequently magnetic beads isolation, using the technique from Miltenyi, this allows a high purity.
[摘要] 这种方法允许你从小鼠脾脏中分离不同的亚类单核细胞,如B细胞,T细胞,CD4 +和CD8 + T。 通过使用来自Miltenyi的技术使细胞与特异性抗体缀合并随后磁珠分离,这允许高纯度。
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