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[流式protocol] 流式细胞术检测记忆性和调节性B细胞

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发表于 2013-4-16 13:38:34 | 显示全部楼层 |阅读模式

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研究B细胞亚群对于免疫治疗和功能相关的研究非常重要。免疫缺陷性疾病、淋巴瘤和自身免疫性疾病均显示了B细胞亚群异常和功能异常。了解B细胞功能和监测B细胞亚群比例的升高或阶段,可为这些疾病的免疫反应监测提供有效的工具。这里提供了两个protocol:一种是检测人类记忆性B细胞,一种是检测小鼠调节性B细胞。
调节性B细胞,即“Bregs”已被大量实验模型证明,其通过分泌IL-10维持外周免疫耐受。
记忆性B细胞在免疫系统中起着重要作用,可对之前已接触过的抗原做出识别和快速反应。
各类B细胞的产生过程

各类B细胞的产生过程

各类B细胞的产生过程


人记忆性B细胞和小鼠调节性B细胞的检测步骤

人记忆性B细胞和小鼠调节性B细胞的检测步骤

人记忆性B细胞和小鼠调节性B细胞的检测步骤


小鼠调节性B细胞结果分析

小鼠调节性B细胞

小鼠调节性B细胞



人记忆性B细胞结果分析

人记忆性B细胞

人记忆性B细胞


===原文摘要=======
Memory and Regulatory B Cell Sub-population Detection by Flow Cytometry
Mark Santos, Wenying Zhang, Roberto Renteria, Jason Whalley, and Matthew Hsu
EMD Millipore, 28820 Single Oak Drive, Temecula, CA 92590 USA

Abstract
Investigating B cell sub-populations is an important area of  research due to its therapeutic relevance and function in the  immune response. Immunodeficiency, lymphoma, and  autoimmune diseases show abnormal B cell subpopulation  and function. Understanding B cell functions and having the  ability to monitor an increase or decrease in B cell subpopulation levels can provide a useful tool for understanding  the immunological response to certain diseases. This  knowledge can provide opportunities to devise therapeutic  strategies to help combat against immunological disorders.  Here we propose two methods for the detection of B cell  subtypes in both human and murine models: one for detection  of human memory B cells and one for identifying mouse  regulatory B cells. In these assays, we have developed  monoclonal antibodies and their conjugates for multi-color  analysis to enable simultaneous detection of these cells.

Introduction
Memory B cells play an integral role in the immune system for their  ability to recognize and react quickly to previously encountered antigens and protect the body from many and future infections.  Originating from lymphocytes that are developed and are activated  in the bone marrow, these cells are committed to respond rapidly when re-exposure to pathogens or other invading substances  occur. By using anti-human CD5, anti-human CD19, and antihuman CD27 monoclonal directly conjugated antibodies, we were  able to determine memory B cell and CD5 + B cells populations, as  well as quantitatively measure the percentage of cells within each  population.
Regulatory B cells are a unique subset of small white blood cells  which produce a potent cytokine, IL-10, a protein that can inhibit  immune responses. B10 cells function to protect host tissues from  immunopathology during infections. Breg subsets can significantly  influence T-cell activation and inflammatory responses through IL- 10 production. Understanding where B10 cells propogate (normally represent 1-3% of the cell population) can be useful information as  B10 cell numbers expand during autoimmunity and acute  inflammation. By performing multi-parametric analysis using CD5,  CD19, and CD1d antibodies by flow cytometry, we are able to  accurately discriminate low frequency and phenotypically unique  subset of regulatory B cells

发表于 2014-4-20 14:23:08 | 显示全部楼层
请教各位高手,人外周血记忆B细胞我看文献中一般用CD19/CD27/IgD联合分析,通过CD27 VS IgD把记忆B细胞分为四类或者五类,这其中的CD27 high plasma cells和CD27 IgD- Switch memory B cell是如何划分这两者之间的界限?

附上一篇相关的参考文献,就是文中的Figure1中的五类区分

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下载PDF: Rheumatology-2010-Moura-1082-92.pdf (573.39 KB, 下载次数: 22, 售价: 1 流星)
 楼主| 发表于 2014-4-20 18:21:08 | 显示全部楼层
dragonhzqsmmu 发表于 2014-4-20 14:23
请教各位高手,人外周血记忆B细胞我看文献中一般用CD19/CD27/IgD联合分析,通过CD27 VS IgD把记忆B细胞分为 ...

感谢贺兄的分享,学习了这种细分的方法,从图中看是根据第Ⅱ群(pre-switch memory B cells)的右边界来定义high和+的,见下图中的红线:

2014-04-20_181755.gif


naı¨ve B cells (I): CD19+IgD+CD27-;
pre-switch memory B cells (II) :CD19+IgD+CD27+;
post-switch memory B cells (III) : CD19+IgD-CD27+;
plasma cells (IV) :CD19+IgD-CD27high.


发表于 2014-4-20 19:31:46 | 显示全部楼层
多谢LZ解答,请您帮忙看看这个结果我的左侧的设门的位置是不是就是这个虚线?

虚线是把右侧的细胞全部压在下面还是怎么弄?我看文献里面在红线的右侧还是有不少细胞的,这个如何来界定??
设门.jpg
 楼主| 发表于 2014-4-20 19:48:09 | 显示全部楼层
dragonhzqsmmu 发表于 2014-4-20 19:31
多谢LZ解答,请您帮忙看看这个结果我的左侧的设门的位置是不是就是这个虚线?

虚线是把右侧的细胞全部压在 ...

你把点图换成等高线图试试
发表于 2014-4-20 20:02:53 | 显示全部楼层
本帖最后由 dragonhzqsmmu 于 2014-4-20 20:49 编辑
niwanmao 发表于 2014-4-20 19:48
你把点图换成等高线图试试

明白了,多谢高手指点啊

但还是有点小问题,诸如下面的左边的分群OK,但右边的如何划分呢?我是根据左侧等高线图的外边第五个圆圈为开放连接右侧细胞群来分界的

另外我下面的naive的左侧界限和上限是否也需要调整?
问题2.jpg
发表于 2014-7-25 21:53:39 | 显示全部楼层
介绍的很详细
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