In vitro Antigen-presentation Assay for Self- and Microbial-derived Antigens
|
Author:
Date:
2017-06-05
[Abstract] Antigen presenting cells (APC) are able to process and present to T cells antigens from different origins. This mechanism is highly regulated, in particular by Patter Recognition Receptor (PRR) signals. Here, I detail a protocol designed to assess in vitro the capacity of APC to present antigens derived from bacteria, apoptotic and infected apoptotic cells.
[摘要] 抗原呈递细胞(APC)能够处理和呈递来自不同来源的T细胞抗原。这种机制是高度调节的,特别是通过Patter Recognition Receptor(PRR)信号。在这里,我详细说明了一种设计用于评估体外的APC方案,用于展示来源于细菌,凋亡和感染的凋亡细胞的抗原。
背景 T细胞淋巴细胞在其表面上表达T细胞受体(TCR),其允许识别作为与主要组织相容性复合物(MHC)分子结合的抗原加工和呈递的抗原的细胞(自身)或微生物(非自身)抗原)呈递细胞(APC)。 APC能够处理抗原并将其呈递给T细胞,并且MHC-TCR相互作用是感染和自身免疫应答期间T细胞活化的关键步骤。  以前的作品已经描述了基于刺激模式识别受体(PRR),例如toll样受体(TLR)(Blander和Medzhitov,2004和2006)的抗原呈递的调节机制。实际上,特异性地来自含有微生物病原体的吞噬体的TLR信号有利于在MHC-II分子内呈递非自身抗原。另一方面,凋亡细胞吞噬后产生的自身抗原由于不存在TLR刺激而导致溶酶体降解。然而,当两者都来自感染的凋亡细胞并且同时由相同的吞噬体携带时,自身和非自身抗原的分离不会发生,其由针对抗原呈递的TLR信号最佳地定制。已经使用骨髓来源的树突状细胞(BMDC)和凋亡性小鼠B细胞 - ...
|
|
Quantification of Tumor Material Uptake
|
Author:
Date:
2016-10-20
[Abstract] Extracellular tumor material including exosomes, microvesicles and apoptotic tumor debris may help cancers invade new organs. Enhancing the removal of extracellular tumor material by immune cells represents a novel immunotherapy approach for preventing cancer metastasis. This protocol quantifies the uptake and removal of extracellular tumor material from circulation and tissues by immune cells. In this assay fluorescent tumor cells are transferred into mice, and then immune cells are quantified by either flow cytometry or imaging cytometry for their uptake of tumor material.
[摘要] 包括外来体,微泡和凋亡性肿瘤碎片的细胞外肿瘤材料可以帮助癌症侵入新器官。增强免疫细胞对细胞外肿瘤材料的去除代表了用于预防癌症转移的新型免疫治疗方法。该方案定量免疫细胞从循环和组织吸收和去除细胞外肿瘤物质。在该测定中,将荧光肿瘤细胞转移到小鼠中,然后通过流式细胞术或成像细胞计量术来定量免疫细胞对肿瘤材料的摄取。 [背景] 研究已经证明,包括从肿瘤脱落的外来体,微泡和凋亡性肿瘤碎片的细胞外肿瘤材料是肿瘤转移,生长和逃避免疫应答的重要介质(Vader等人,2014; Pucci和Pittet ,2013; Pucci等人,2016)。免疫细胞具有去除,应答和转运这种循环肿瘤材料的能力(Hanna等人,2015; Pucci等人,2016; Headley等人。,2016)。该协议提供了一种新的方法来量化特定免疫细胞群体的肿瘤材料摄取,并且可以适于测试调节肿瘤材料摄取的免疫靶标。该协议可以帮助更好地理解对细胞外肿瘤材料的免疫应答,希望最终开发针对癌症发展中的细胞外肿瘤材料的新疗法。
|
|
Isolation and Purification of Murine Microglial Cells for Flow Cytometry
|
Author:
Date:
2016-01-05
[Abstract] The detailed protocol is used to isolate different cell types from murine brain as glial cells, including microglia, using an enzymatic digestion that minimizes cellular mortality. A Percoll gradient (30% to 80%) separation allows a maximal recovery of isolated murine microglial cells prior to flow cytometry analysis.
[摘要] 详细的协议是用于分离不同的细胞类型从鼠脑作为神经胶质细胞,包括小胶质细胞,使用最小化细胞死亡率的酶消化。 Percoll梯度(30%至80%)分离允许在流式细胞术分析之前分离的小鼠胶质细胞的最大恢复。
|
|