| Isolation and Culture of Human Adipose-derived Stem Cells from Subcutaneous and Visceral White Adipose Tissue Compartments
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Author:
Date:
2016-11-20
[Abstract] Human Adipose-derived Stem/Stromal Cells (ASCs) have been widely used in stem cell and obesity research, as well as clinical applications including cell-based therapies, tissue engineering and reconstruction. Compared with mesenchymal stem cells (MSCs) derived from other tissues such as umbilical cord and bone marrow, isolation of ASCs from human white adipose tissue (WAT) has great advantages due to its rich tissue source and simple surgical procedure. In this detailed protocol we describe a protocol to isolate and characterize ASCs from human WAT. Molecular characterization of isolated ASCs ...
[摘要] Human Adipose-derived Stem/Stromal Cells (ASCs) have been widely used in stem cell and obesity research, as well as clinical applications including cell-based therapies, tissue engineering and reconstruction. Compared with mesenchymal stem cells (MSCs) derived from other tissues such as umbilical cord and bone marrow, isolation of ASCs from human white adipose tissue (WAT) has great advantages due to its rich tissue source and simple surgical procedure. In this detailed protocol we describe a protocol to isolate and characterize ASCs from human WAT. Molecular characterization of isolated ASCs ...
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| Cell-based Assays to Monitor AID Activity
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Author:
Date:
2016-02-05
[Abstract] The enzyme Activation induced deaminase (AID) underpins antibody affinity maturation and isotype switching through its mutagenic activity of deaminating deoxycytidine to deoxyuridine in DNA. Subsequent processing of the deoxyuridine initiates the processes of somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. Structure-function analysis of AID requires sensitive and biologically relevant methods to measure its various activities. Here we describe simple but effective methods to measure 1) the ability of AID to mutate the Escherichia coli genome, which ...
[摘要] 酶活化诱导的脱氨酶（AID）通过其将脱氧胞苷脱氨基到DNA中的脱氧尿苷的诱变活性来支持抗体亲和力成熟和同种型转换。脱氧尿苷的后续加工引发B细胞中体细胞超突变（SHM）和类型转换重组（CSR）的过程。 AID的结构功能分析需要灵敏和生物相关的方法来测量其各种活动。在这里我们描述简单但有效的方法来测量1）AID突变大肠杆菌基因组的能力，其提供其催化活性的指示; 2）AID通过补充DT40鸡B细胞系的衍生物来进行SHM的能力; 3）AID通过补充AID缺乏的原代小鼠B细胞来进行CSR的能力。三种方法的组合，伴随着AID亚细胞定位和蛋白质表达水平和稳定性的必要分析作为对照，允许AID的详细结构功能研究。
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| Fluorescence Microscopy Analysis of Drug Effect on Autophagosome Formation
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Author:
Date:
2014-04-05
[Abstract] The autophagy protein, LC3 represents a reliable characteristic marker for autophagosomal structures. The initial LC3 is processed by the cysteine protease autophagy-related gene 4 (Atg4) at its C terminus in order to create LC3-I generally localized in the cytoplasm. Afterwards LC3-I is conjugated with phosphatidylethanolamine (PE) to become LC3-PE or LC3-II predominantly localised on the autophagosomal membranes (outer and inner). Autolysosomal content of LC3-II is very low as upon autophago/lysosomal fusion it is either cleaved off from the outer membrane by Atg4 or degraded together with ...
[摘要] 自噬蛋白，LC3代表自噬体结构的可靠特征标记。最初的LC3由半胱氨酸蛋白酶自噬相关基因4（Atg4）在其C末端处理，以产生通常位于细胞质中的LC3-I。之后LC3-I与磷脂酰乙醇胺（PE）缀合以变成主要定位在自噬体膜（外部和内部）上的LC3-PE或LC3-II。 LC3-II的自溶酶体内含物非常低，因为在自噬/溶酶体融合时，其被Atg4从外膜切割或与内膜一起通过溶酶体活性降解。因此，GFP-LC3和mCherry-GFP-LC3可能通过常规或共聚焦荧光显微镜（FM）可视化。在这种情况下，mCherry-GFP-LC3或GFP-LC3细胞质池被可视化为均匀分散的信号，并且含有mCherry-GFP-LC3-II或GFP-LC3-II的自噬体被检测为点状形成。斑点数可以用作自噬体丰度的标志物。一般来说，我们建议计数每个细胞的GFP-LC3斑点的平均数。
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