Isolation, Culture, and Maintenance of Mouse Intestinal Stem Cells
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Author:
Date:
2016-02-20
[Abstract] In this protocol we describe our modifications to a method to isolate, culture and maintain mouse intestinal stem cells as crypt-villus forming organoids. These cells, isolated either from the small or large intestine, maintain self-renewal and multilineage differentiation potential over time. This provides investigators a tool to culture wild type or transformed intestinal epithelium, and a robust assay for stem cell tissue homeostasis in vitro.
[摘要] 在这个协议,我们描述我们修改分离,培养和维持鼠肠干细胞作为隐窝 - 绒毛形成类器官的方法。 从小肠或大肠分离的这些细胞随时间保持自我更新和多向分化潜能。 这为研究人员提供了培养野生型或转化的肠上皮的工具,以及用于体外干细胞组织稳态的稳健试验。
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Colony Immunoblotting Assay for Detection of Bacterial Cell-surface or Extracellular Proteins
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Author:
Date:
2013-09-05
[Abstract] This simple protocol describes how to detect antigens from agar-grown bacterial colonies transferred to nitrocellulose using specific antibodies. The protocol is well suitable for detection of bacterial proteins exposed on the cell surface or secreted to the extracellular space and it can be modified also for detection of intracellular proteins. The assay can distinguish bacterial clones with different expression rates (high, medium and low) from colonies that do not express target protein. We used this assay for screening of Mat fimbriae-producing Escherichia coli mutants obtained by mini-Tn5 transposon mutagenesis and immunomagnetic separation (Lehti et al., 2013).
[摘要] 这个简单的协议描述如何使用特异性抗体检测从转移到硝酸纤维素的琼脂生长的细菌菌落的抗原。 该方案非常适合于检测暴露于细胞表面或分泌至细胞外空间的细菌蛋白,并且其还可以被修饰以用于检测细胞内蛋白。 该测定可以区分具有不同表达率(高,中和低)的细菌克隆与不表达靶蛋白的集落。 我们使用该测定法用于筛选通过mini-Tn5转座子诱变和免疫磁性分离获得的产生Mat菌毛的大肠杆菌突变体(Lehti等人,2013)。
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In vitro Culture of Human PBMCs
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Author:
Date:
2013-02-05
[Abstract] Peripheral blood mononuclear cells (PBMCs) consist of chiefly of lymphocytes and monocytes. Purified PBMCs are used in vitro to evaluate a variety of functions of lymphocytes viz; a) proliferation to mitogenic (PHA, Con-A) stimulation, b) monitoring of prior sensitisation in antigen recall assays by scoring lymphocyte proliferation, c) immunophenotyping for surface markers as well as intracellular molecules in monocytes and lymphocytes etc. Activation of monocytes/macrophages by small molecules, cytokines and pathogen components can also be monitored. PBMCs can also be used for a variety of structural and functional studies for addressing issues in human immunology such as scoring for apoptosis and production of cytokines as well as other mediators in vitro.
[摘要] 外周血单核细胞(PBMC)主要由淋巴细胞和单核细胞组成。 纯化的PBMC在体外用于评价淋巴细胞的多种功能, a)增殖至促有丝分裂(PHA,Con-A)刺激,b)通过评估淋巴细胞增殖来监测抗原召回试验中的先前致敏,c)表面标志物以及单核细胞和淋巴细胞等中的细胞内分子的免疫表型。 也可以监测小分子,细胞因子和病原体组分的巨噬细胞。 PBMC还可以用于多种结构和功能研究,用于解决人免疫学中的问题,例如细胞凋亡评分和细胞因子以及其他介体的体外生产。
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