| Killer Cell Ig-like Receptors (KIR)-Binding Assay for Tumor Cells
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Author:
Date:
2016-09-20
[Abstract] Natural killer (NK) cells play key roles in innate and adaptive immune responses against virus and tumor cells. Their function relies on the dynamic balance between activating and inhibiting signals through receptors that bind ligands expressed on target cells. The absence of inhibitory receptor engagement with their ligands and the presence of activating signals transmitted by activating receptors interacting with specific ligands, leads to NK cell activation (Lanier, 2005; Raulet et al., 2001). Thus, the balance of the ligands expressed for inhibitory and activating receptors determines whether NK cells will become activated to kill the target cells. This protocol allows to assign a precise ligand specificity to any given receptor on NK cells. Thus, if a tumor cell expresses ...
[摘要] 自然杀伤（NK）细胞在针对病毒和肿瘤细胞的先天和适应性免疫应答中发挥关键作用。它们的功能依赖于通过结合靶细胞上表达的配体的受体在激活和抑制信号之间的动态平衡。不存在与其配体的抑制性受体接合和通过活化受体与特异性配体相互作用传递的激活信号的存在导致NK细胞活化（Lanier，2005; Raulet等人，2001）。因此，针对抑制性和激活受体表达的配体的平衡决定了NK细胞是否将被激活以杀死靶细胞。该协议允许给NK细胞上的任何给定受体分配精确的配体特异性。因此，如果肿瘤细胞表达配体，则该方案将允许评价其与特异性受体的相互作用。特别地，通过与HLA I类重链残基和结合肽的氨基酸残基的直接接触，杀伤细胞免疫球蛋白（Ig）样受体（KIR）识别它们的配体（HLA I类分子）。该方案将允许测试氨基酸取代或其他突变对KIR与HLA I型结合的影响。我们使用该方案描述ERAP1（MHC I类抗原加工的关键组分）在调节NK细胞中的作用通过控制抑制性受体的接合来控制细胞功能（Cifaldi等人，2015）。
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| Peptide Loading on MHC Class I Molecules of Tumor Cells
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Author:
Date:
2016-09-20
[Abstract] MHC class I molecules present peptides to cytotoxic T cells allowing the immune system to scan for intracellular pathogens and mutated proteins. The generation of antigenic peptides is a multistep process that ends in the endoplasmic reticulum (ER). Only peptides with the right length and sequence will bind nascent MHC class I molecules in the ER. This protocol allows for detachment of the endogenous peptides bound to MHC class I molecules by preserving them for the binding of high affinity synthetic peptides. The complete dissociation of endogenous peptides by mild acid treatment as well as the binding of synthetic peptides to MHC class I molecules will be evaluated measuring HLA class I molecules express on the cell surface by flow cytometry. The mouse antibody W6/32 which recognizes ...
[摘要] MHC I类分子将肽呈递到细胞毒性T细胞，允许免疫系统扫描胞内病原体和突变蛋白。抗原肽的产生是以内质网（ER）结束的多步过程。只有具有正确长度和序列的肽将结合ER中的新生MHC I类分子。该方案允许通过保持结合MHC I类分子的内源肽与高亲和力合成肽的结合而脱离。通过温和酸处理以及合成肽与MHC I类分子的结合来评价内源肽的完全解离，通过流式细胞术测量在细胞表面上表达的HLA I类分子。识别β2m相关HLA-A，-B，-C，-E和-G重链的小鼠抗体W6/32适合于此提议。表达表面HLA I类分子的任何肿瘤细胞系适于测定。另一个重要方面是知道肿瘤细胞系的HLA I类分型以允许选择已知的高亲和力肽。
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| Severe Fever with Thrombocytopenia Syndrome Virus Infection of Cell Cultures
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Author:
Date:
2014-10-20
[Abstract] Severe fever with thrombocytopenia syndrome virus (SFTSV) can infect multiple cells, such as Vero, RD, BHK21, HUVEC and 293T. Vero cells are highly susceptible, so they were used to isolate virus from sera of patients, as well as for producing virus in the laboratory. Other cells can be used for additional functional analysis.
[摘要] 血小板减少综合征病毒（SFTSV）的严重发热可以感染多种细胞，例如Vero，RD，BHK21，HUVEC和293T。 Vero细胞是高度易感的，因此它们用于从患者的血清中分离病毒，以及用于在实验室中产生病毒。 其他细胞可用于额外的功能分析。
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