| Isolation and Culture of Human CD133+ Non-adherent Endothelial Forming Cells
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Author:
Date:
2016-04-05
[Abstract] Circulating endothelial progenitor cells (EPCs) have been the focus of many clinical trials due to their roles in revascularisation following ischemic events such as acute myocardial infarction as well as their contribution to vascular repair during organ transplantation. Research on EPCs has been controversial due to the lack of distinct markers expressed at the cell surface and varying methods for isolation and culture have resulted in the identification of a multitude of cell types, with differing phenotype and function, all falling under the label of “EPCs”. The most widely documented EPCs isolated for cell therapy are adherent in nature and lacking the progenitor markers such as CD133 and therefore unlikely to represent a true circulating EPC, the cells mobilised in response to a ...
[摘要] 循环内皮祖细胞（EPCs）已经成为许多临床试验的焦点，因为它们在缺血事件例如急性心肌梗死后的血管再形成中的作用以及它们在器官移植期间对血管修复的贡献。由于缺乏在细胞表面表达的不同标记物，EPCs的研究已经引起争议，并且用于分离和培养的不同方法导致鉴定了具有不同表型和功能的多种细胞类型，所有这些都属于" EPCs"。分离用于细胞治疗的最广泛记载的EPCs本质上是粘附的，缺乏祖细胞标志物例如CD133，因此不太可能代表真正的循环EPC，所述细胞响应于血管损伤而动员。
我们最近发布了非粘附内皮形成细胞（naEFCs）群体的分离和广泛表征（Appleby等人，2012）（图1）。这些细胞与成熟内皮细胞标记（VEGFR2，CD144和CD31）一起表达祖细胞标记（CD133，CD34，CD117，CD90和CD38）。这些细胞还表达低水平的CD45，但不表达将其与"早期"EPC区分开的淋巴标志物（CD3，CD4，CD8）或骨髓标志物（CD11b和CD14），"晚期生长EPC"[最近称为内皮细胞集落形成细胞（ECFCs）]以及成熟内皮细胞（ECs）。图2A例示了naEFC的表面表达谱。功能研究证明这些naEFC（i）结合于玻璃体凝集素（图2A），（ii）显示乙酰化低密度脂蛋白摄取，（iii）增加血管细胞粘附分子（VCAM-1）响应于肿瘤坏死因子的表达和（iv）与成熟EC的共培养增加了在三维体外基质中的管，小管分支和环的数目。更重要的是，放置在体内的naEFC产生了包含由表达CD144的人EC内衬的脉管系统的新腔，并且有助于科学知识的各种进步（Appleby等人，2012; ...
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| 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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| Ex vivo Natural Killer Cell Cytotoxicity Assay
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Author:
Date:
2013-08-20
[Abstract] Natural Killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. Immunosurveillance of the host by NK cells for malignant and virally-infected cells results in direct cytotoxicity and the production of cytokines to enhance the immune response. This protocol will describe the “gold standard” chromium release assay for measuring the target cell killing capacity of NK cells. Key features of this cytotoxicity assay are that it is performed with sorted NK cells as the effectors and any Major Histocompatibility Class I (MHC-I)-low or deficient tumor cell line can be used as the target cells.
[摘要] 天然杀伤（NK）细胞是细胞毒性淋巴细胞，其构成先天性免疫系统的主要组分。 通过NK细胞对恶性和病毒感染的细胞的宿主的免疫监视导致直接的细胞毒性和细胞因子的产生，以增强免疫应答。 该方案将描述用于测量NK细胞的靶细胞杀伤能力的"金标准"铬释放测定。 该细胞毒性测定的主要特征是其使用分选的NK细胞作为效应物进行，任何主要组织相容性I类（MHC-1） - 低或缺陷的肿瘤细胞系可以用作靶细胞。
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