| Evaluation of Cross-presentation in Bone Marrow-derived Dendritic Cells in vitro and Splenic Dendritic Cells ex vivo Using Antigen-coated Beads
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Author:
Date:
2016-11-20
[Abstract] Antigen presentation by MHC class I molecules, also referred to as cross-presentation, elicits cytotoxic immune responses. In particular, dendritic cells (DC) are the most proficient cross-presenting cells, since they have developed unique means to control phagocytic and degradative pathways.
This protocol allows the evaluation of antigen cross-presentation both in vitro (by using bone marrow-derived DC) and ex vivo (by purifying CD8+ DC from spleen after incorporation of particulate antigen) using ovalbumin (OVA)-coupled particles. Cross-presentation efficiency is measured by three different readouts: the B3Z hybridoma T cell line (Karttunen et al., 1992) and stimulation of antigen-specific CD8+ T cells (OT-I) (Kurts et al ...
[摘要] 通过MHC I类分子的抗原呈递，也称为交叉呈递，引起细胞毒性免疫应答。特别地，树突细胞（DC）是最熟练的交叉呈递细胞，因为它们已经开发了控制吞噬和降解途径的独特手段。 <此协议允许在体外（通过使用骨髓衍生的DC）和离体（通过纯化CD8 + ）评价抗原交叉呈递。 （OVA）偶联的颗粒后，来自脾脏的DC/DC结合颗粒抗原）。通过三种不同的读数测量交叉呈递效率：B3Z杂交瘤T细胞系（Karttunen等人，1992）和抗原特异性CD8 + T细胞的刺激OT-I）（Kurts等人，1996），在用羧基荧光素琥珀酰亚胺酯（CFSE）标记它们之后分析CD69表达或OT-I增殖的OT-I活化。通过使用这种方法，我们可以最近显示DCs能够在TLR4结合时瞬时增加交叉表达效率（Alloatti等人，2015）。 [背景] 在小鼠中，抗原呈递细胞（APC）能够吸收外源抗原以加工它们，并将源自这些外源抗原的肽加载到主要组织相容性复合体（MHC）I类分子上。肽-MHC I复合物随后被转运到质膜，在那里它们可以呈递到CD8 + T细胞，从而促进T细胞活化，这被称为交叉呈递（Joffre等人， al 。，2012）。在不同的APC中，树突状细胞（DC）在交叉呈递方面表现优异，并且包含表达XCR1标记的不同亚群，其已经显示非常有效地交叉呈递抗原（即CD8 + 来自脾的DC和来自皮肤和肺的CD103 ...
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| HIV-1 Single Cycle Infection
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Author:
Date:
2013-04-05
[Abstract] The role of a viral or cellular protein on HIV-1 infection can sometimes be difficult to assess in a system where the virus is able to replicate for several cycles. Indeed, some effects are only observable at early time points, and can be masked (or on the contrary, artificially increased) after several rounds of viral replication. Therefore, to clearly know at which step of HIV-1 replication cycle one protein acts, it is important to be able to study one cycle of infection only. This protocol allows rapid and robust quantification of HIV-1 single cycle infection, and can be used to compare mutant viruses, or treatments with different drugs.
[摘要] 病毒或细胞蛋白质对HIV-1感染的作用有时可能难以在其中病毒能够复制几个周期的系统中评估。 事实上，一些作用仅在早期时间点可观察到，并且可以在几轮病毒复制后被掩蔽（或相反，人为增加）。 因此，为了清楚知道一种蛋白质作用于HIV-1复制循环的哪个步骤，重要的是能够仅研究一个周期的感染。 该协议允许快速和强大的量化HIV-1单周期感染，并可用于比较突变病毒或不同药物的治疗。
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| In vitro Ag Cross-presentation and in vivo Ag Cross-presentation by Dendritic Cells in the Mouse
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Author:
Date:
2012-12-20
[Abstract] Antigen cross presentation is important for effective immune responses to tumors and viral infections. Dendritic cells are professional antigen presenting cells and are unique in their ability to cross-present exogenous antigens on MHC class I molecules and activate antigen specific cytotoxic T cells. This protocol describes antigen cross presentation by dendritic cells (DCs) (bone marrow derived DCs and splenic DCs) in an in vitro and in an in vivo assay system using soluble ovalbumin protein.
[摘要] 抗原交叉呈递对于对肿瘤和病毒感染的有效免疫应答是重要的。 树突状细胞是专业抗原呈递细胞，并且在其将MHC I类分子上的外源抗原交叉呈递并激活抗原特异性细胞毒性T细胞的能力方面是独特的。 该方案描述了在体外和在体内使用可溶性卵白蛋白蛋白质的体系中通过树突细胞（DC）（骨髓来源的DC和脾DC）的抗原交叉呈递 。
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