Isolation and Primary Culture of Adult Mouse Cardiac Fibroblasts
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Author:
Date:
2016-07-05
[Abstract] Fibroblasts are often used as a feeder layer for progenitor or stem cells in co-culture systems. In the heart fibroblasts are important for cardiac development, homeostasis, and remodelling. They provide cardiomyocytes and progenitor cells not only with nutrition but also secrete extracellular matrix that forms the microenvironment that ensures cell survival and function. Although different kinds of mouse fibroblasts have been used in co-cultures (embryonic, skin and cardiac fibroblasts) adult mouse cardiac fibroblasts (AMCFs) create the closest microenvironment to the adult murine heart for culturing adult mouse cardiac progenitor cells. This protocol describes the isolation of cardiac fibroblasts from adult mouse hearts as well as their maintenance in culture.
[摘要] 成纤维细胞通常用作共培养系统中的祖细胞或干细胞的饲养层。 在心脏成纤维细胞对于心脏发育,体内平衡和重塑是重要的。 他们提供心肌细胞和祖细胞不仅与营养,而且分泌细胞外基质,形成微环境,确保细胞的生存和功能。 尽管不同种类的小鼠成纤维细胞已用于共培养(胚胎,皮肤和心脏成纤维细胞),但是成年小鼠心脏成纤维细胞(AMCFs)为培养成年小鼠心脏祖细胞产生了与成年鼠心脏最接近的微环境。 该协议描述了从成年小鼠心脏分离心脏成纤维细胞以及它们在培养物中的维持。
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In vivo OVA-specific Cytotoxic CD8+ T Cell Killing Assay
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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。
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In vitro Tumor Cell Migration Assay Using ThinCertsTM (Transwells)
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Author:
Date:
2016-06-05
[Abstract] The high migration rate of tumor cells often results in poor prognosis for the survival of the patients. Here, we describe a protocol to measure the migration of cells using a quantitative assay. The relative tumor cell migration was measured using ThinCertsTM cell culture inserts and a lactate dehydrogenase (LDH) assay to quantify the relative cell number. The quantification of the migration with the LDH kit is much more precise than other methods using i.e. crystal blue to count the cells.
[摘要] 肿瘤细胞的高迁移率通常导致患者的生存的不良预后。 在这里,我们描述了使用定量测定测量细胞迁移的协议。 使用ThinCerts TM细胞培养插入物和乳酸脱氢酶(LDH)测定来测量相对肿瘤细胞迁移以定量相对细胞数。 使用LDH试剂盒的迁移的定量比使用结晶蓝计数细胞的其它方法精确得多。
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