| 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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| In vivo Lineage-tracing Studies in a Cancer Stem Cell Population in Neuroblastoma
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Author:
Date:
2014-04-20
[Abstract] Tumors are comprised of heterogeneous subpopulations that may exhibit differing capacity for differentiation, self-renewal, and tumorigenicity. In vivo lineage-tracing studies are a powerful tool for defining the role of tumor subpopulations in tumor growth and as targets for therapeutic agents. This protocol describes using a neuroblastoma cancer cell line transduced with two different fluorescent proteins (GFP and tdTomato) to track the specific contributions of cells expressing the GCSF receptor (CD114+) or not (CD114-) on tumor growth in vivo.
[摘要] 肿瘤由异质亚群组成，其可以表现出不同的分化，自我更新和致瘤性能力。 体内谱系追踪研究是定义肿瘤亚群在肿瘤生长中的作用和作为治疗剂靶标的有力工具。 该方案描述了使用用两种不同的荧光蛋白（GFP和tdTomato）转导的神经母细胞瘤癌细胞系来追踪表达GCSF受体（CD114 + sup）+或CD114（sup）+的细胞的特异性作用 ）对肿瘤生长的作用。
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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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