| Exit from Pluripotency Assay of Mouse Embryonic Stem Cells
|
|
Author:
Date:
2017-08-20
[Abstract] A novel method to assess the dissolution of the core pluripotency transcription-factor circuit of mouse Embryonic Stem Cells (mESCs) has been developed (Ying et al., 2003; Betschinger et al., 2013). In order to efficiently identify genes essential for the break-down of the pluripotency network in mutant mESCs with proliferation defects, we adapted this ‘exit from pluripotency assay’ (Bodak et al., 2017; Cirera-Salinas et al., 2017). The protocol described here has been successfully applied to several mESC lines and is easily transposable from one laboratory to another.
[摘要] 已经开发了评估小鼠胚胎干细胞（mESCs）的核心多能转录因子电路溶解的新方法（Ying等，2003; Betschinger等，2013）。 为了有效识别具有增殖缺陷的突变体mESCs中多能网络分解所必需的基因，我们调整了这种“多能性测定法”（Bodak等，2017; Cirera-Salinas等，2017）。 这里描述的方案已经成功应用于几个mESC系列，并且可以容易地从一个实验室转座到另一个实验室。
【背景】几十年来，科学家已经尝试确定基因与一般（例如胚胎体）或定向（例如，神经元前体细胞）分化方案的mESCs的分化潜能的机制。最近，发现2i培养基允许在体外俘获天真的干细胞（Ying et al。，2008）。 ...
|
|
|
| Differentiation of Human Induced Pluripotent Stem Cells (iPS Cells) and Embryonic Stem Cells (ES Cells) into Dendritic Cell (DC) Subsets
|
|
Author:
Date:
2017-08-05
[Abstract] Induced pluripotent stem cells (iPS cells) are engineered stem cells, which exhibit properties very similar to embryonic stem cells (ES cells; Takahashi and Yamanaka, 2016). Both iPS cells and ES cells have an extraordinary self-renewal capacity and can differentiate into all cell types of our body, including hematopoietic stem/progenitor cells and dendritic cells (DC) derived thereof. This makes iPS cells particularly well suited for studying molecular mechanisms of diseases, drug discovery and regenerative therapy (Grskovic et al., 2011; Bellin et al., 2012; Robinton and Daley, 2012).
DC are the major antigen presenting cells of the immune system and thus they are key players in modulating and directing immune responses (Merad et al., 2013). DC ...
[摘要] 诱导的多能干细胞（iPS细胞）是工程干细胞，其表现出与胚胎干细胞（ES细胞，Takahashi和Yamanaka，2016）非常相似的性质。 iPS细胞和ES细胞都具有非凡的自我更新能力，可以分化成我们身体的所有细胞类型，包括造血干细胞/祖细胞和源自其的树突状细胞（DC）。这使得iPS细胞特别适用于研究疾病，药物发现和再生治疗的分子机制（Grskovic等人，2011; Bellin等人，2012; Robinton和Daley，2012）。
＆NBSP; DC是免疫系统的主要抗原呈递细胞，因此它们是调节和引导免疫应答的关键参与者（Merad等人，2013）。 DC巡逻外周和界面组织（例如，肺，肠和皮肤）以检测入侵的病原体，并且在激活时，它们迁移到淋巴结以激活和引发淋巴细胞。
＆NBSP; DC包含具有功能专门子集的表型异质家族（Schlitzer和Ginhoux，2014）。通常，经典DC（cDC）和浆细胞样DC（pDC）是分别表现出典型的和等离子体细胞样的DC形态。 cDC识别许多病原体并在激活后分泌促炎细胞因子，而pDC专门检测细胞内病原体并分泌I型干扰素（Merad等，2013; Schlitzer和Ginhoux，2014）。在被称为CD141 Clec9a + cDC1和CD1c + ...
|
|
|
| In vivo OVA-specific Cytotoxic CD8+ T Cell Killing Assay
|
|
Author:
Date:
2016-06-20
[Abstract] Cytotoxic CD8+ T cells are responsible for the lysis of cells expressing peptides associated with MHC class I molecules and derived from infection with a pathogen or from mutated antigens. In order to quantify in vivo this antigen-specific CD8+ T cell killing activity, we use the in vivo killing assay (IVK). Here we describe the protocol for the lysis of cells expressing a CD8+ T cell epitope of the OVA protein (SIINFEKL). Mice are previously immunized with the OVA protein and 7 days after immunization, they receive a mix of target cells, prepared from naive C57BL/6 spleen cells pulsed with the SIINFEKL peptide and labeled with high level of CFSE and of non-pulsed control cells labeled with low level of CFSE. One day later, the spleen ...
[摘要] 细胞毒性CD8 + T细胞负责裂解表达与MHC I类分子相关并且源自病原体感染或突变抗原的肽的细胞。为了在体内定量该抗原特异性CD8 + T细胞杀伤活性，我们使用体内杀伤试验（IVK）。在这里，我们描述了用于裂解表达OVA蛋白（SIINFEKL）的CD8 + T细胞表位的细胞的方案。小鼠先前用OVA蛋白免疫，并且在免疫后7天，它们接受从用SIINFEKL肽脉冲并用高水平的CFSE标记的天然C57BL/6脾细胞制备的靶细胞和标记的非脉冲对照细胞的混合物具有低水平的CFSE。一天后，分离受体小鼠的脾细胞并通过FACS分析以测量CFSE高度细胞和CFSE低度细胞的量。通过在免疫的和未免疫的小鼠中CFSE高与低之间的差计算裂解的百分比。
测量抗原特异性CD8 + T细胞在体内裂解其抗原的能力对评价诱导针对病原体或肿瘤抗原的适应性细胞毒性应答是非常重要的。
|
|
|