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RPMI-1640 Medium

RPMI-1640培养基

Company: Sigma-Aldrich
Catalog#: R8758
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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 - ...

Efficient Production of Functional Human NKT Cells from Induced Pluripotent Stem Cells − Reprogramming of Human Vα24+iNKT Cells
Author:
Date:
2017-05-20
[Abstract]  Antigen-specific T cell-derived induced pluripotent stem cells (iPSCs) have been shown to re-differentiate into functional T cells and thus provide a potential source of T cells that could be useful for cancer immunotherapy. Human Vα24+ invariant natural killer T (Vα24+iNKT) cells are subset of T cells that are characterized by the expression of an invariant Vα24-Jα18 paired with Vβ11, that recognize glycolipids, such as α-galactosylceramide (α-GalCer), presented by the MHC class I-like molecule CD1d. Vα24+iNKT cells capable of producing IFN-γ are reported to augment anti-tumor responses, which affects both NK cells and CD8+ cytotoxic T lymphocytes to eliminate MHC- and MHC+ tumor cells, respectively. Here we describe a ... [摘要]  抗原特异性T细胞来源的诱导多能干细胞(iPSCs)已显示重新分化为功能性T细胞,从而提供可用于癌症免疫治疗的T细胞的潜在来源。不变性自然杀伤T(Vα24 + iNKT)细胞的人Vα24 + 细胞是T细胞的子集,其特征在于与Vβ11配对的不变Vα24-Jα18的表达,其识别糖脂,如α-半乳糖神经酰胺(α-GalCer),由MHC I类分子CD1d呈递。据报道能够产生IFN-γ的Vα24 + i / KT细胞增加抗肿瘤反应,其影响NK细胞和CD8 +细胞毒性T淋巴细胞以消除MHC - 和MHC + 肿瘤细胞。在这里,我们描述了将人Vα24 + iNKT细胞重编程到iPSC中的鲁棒方案,然后将其重新分化为Vα24 + iNKT细胞(iPS-Vα24功能的iNKT)。我们进一步提供了测定iPS-Vα24 + iNKT细胞活性的方案。背景 以前有报道说,针对晚期非小细胞肺癌(NSCLC)和头颈部癌症的Vα24 + iNKT细胞癌免疫治疗的临床试验显示疗效,耐受性良好(Motohashi et al。等人,2009; Yamasaki等人,2011)。然而,已知来自外周血单核细胞(PBMC)的Vα24 ...

Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9
Author:
Date:
2017-04-05
[Abstract]  CRISPR-Cas9 based knockout strategies are increasingly used to analyze gene function. However, redundancies and overlapping functions in biological signaling pathways can call for generating multi-gene knockout cells, which remains a relatively laborious process. Here we detail the application of multi-color LentiCRISPR vectors to simultaneously generate single and multiple knockouts in human cells. We provide a complete protocol, including guide RNA design, LentiCRISPR cloning, viral production and transduction, as well as strategies for sorting and screening knockout cells. The validity of the process is demonstrated by the simultaneous deletion of up to four programmed cell death mediators in leukemic cell lines and patient-derived acute lymphoblastic leukemia xenografts, in which ... [摘要]  基于CRISPR-Cas9的敲除策略越来越多地用于分析基因功能。然而,生物信号通路中的冗余和重叠功能可能需要产生多基因敲除细胞,这仍然是一个相对费力的过程。在这里,我们详细介绍了多色LentiCRISPR载体在人体细胞中同时产生单次和多次敲除的应用。我们提供了一个完整的方案,包括指导RNA设计,LentiCRISPR克隆,病毒生产和转导,以及排序和筛选敲除细胞的策略。该过程的有效性通过同时删除白血病细胞系中多达四个程序性细胞死亡介质和来自患者来源的急性淋巴细胞白血病异种移植物,其中单细胞克隆是不可行的。该协议允许任何具有基本细胞生物学设备的实验室,生物安全2级设备和荧光激活细胞分选功能,可在一个月内有效产生单基因和多基因敲除细胞系或原代细胞。

从对细菌基因组中被称为聚簇定期交织的短回文重复(CRISPR)的遗传元件的好奇的初步观察开始(Ishino等人,1987; Mojica等人,2000 )和随后在哺乳动物细胞中的基因编辑(Cong等人,2013; Mali等人,2013),CRISPR-Cas9已经成为廉价和有效的基因编辑。随着从烟草植物细胞到斑马鱼和原代人类细胞(Hsu等人,2014)的细胞系统的成功应用,CRISPR-Cas9可以通过短的20个核苷酸RNA序列的设计来引导在大基因组内的靶向DNA双链断裂(DSB)(Park等人,2016)。 ...

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