• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Journal of Immunological Methods >Flow cytometric analysis of T cell proliferation in a mixed lymphocyte reaction with dendritic cells.
【24h】

Flow cytometric analysis of T cell proliferation in a mixed lymphocyte reaction with dendritic cells.

机译：与树突状细胞混合淋巴细胞反应中T细胞增殖的流式细胞仪分析。

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

BACKGROUND: Dendritic cells (DCs) are the most potent antigen-presenting cells. They can be generated in vitro from CD14+ cells, and also from CD34+ progenitor cells. Although T cell proliferation using [3H] thymidine incorporation assay has been used widely to check DC function, this technique only provides limited information about the T cell proliferation. Here, we describe a novel method for quantitative analysis of T cell proliferation using flow cytometry. MATERIALS AND METHODS: DCs were generated from CD14+ cells from six healthy blood donors. Monocytes were isolated using positive selection with magnetic cell sorting (MACS) and then cultured with IL-4, GM-CSF, IL-1beta, IL-6, TNF-alpha and PGE(2) to yield fully mature DCs. Allogeneic naive T lymphocytes with known mismatches in HLA classes I and II were cocultured with DCs. Naive T cells without DC stimulation served as negative controls. T cells were harvested on days 0, 3, 5, 7, 9, 11 and analysed by flow cytometry. CD3-ECD and CD4-fluorescein isothiocyanate (FITC) or CD8-FITC antibodies were used to distinguish T cell subsets, whereas T cell activation was measured by assessment of HLA-DR, CD45RO, CD25 and CD71 expression. For T cell quantification, fluorescent microparticles were used. Dead cells were excluded with 7-AAD. The bromdeoxyridine (BrdU)-incorporation ELISA procedure was also performed in order to compare with the T cell proliferation assay with regard to absolute cell counts and CD71 expression. RESULTS: The initial T cell concentration on day 1 was 203.9+/-39.7 (173-265) CD3+/CD4+ cells/micro l and 184.5+/-41.6 (148-260) CD3+/CD8+ cells/micro l. The maximal T cell proliferation was recorded on day 7 with a five- to tenfold T cell expansion which resulted in 1994.9+/-383 (1446-2404) CD3+/CD4+ cells/micro l and 944+/-303.7 (560-1483) CD3+/CD8+ cells/micro l. Furthermore, activation markers of both cell lineages were upregulated and reached maxima on days 7 (CD71) and 9 (CD25, HLA-DR). T cell count/micro l as well as CD71 expression both correlated significantly with BrdU incorporation. CONCLUSION: Flow cytometric analysis permits simple, precise and rapid quantification of T cell proliferation in a mixed lymphocyte reaction with DCs. Activation, proliferation and cell viability can be simultaneously determined. CD71 is particularly well suited as an activation marker for the simultaneous measurement of T cell proliferation. Thus, specific T cell subsets involved in antigen-specific proliferation can be evaluated in detail.
机译：背景：树突状细胞（DC）是最有效的抗原呈递细胞。它们可以从CD14 +细胞以及CD34 +祖细胞体外产生。尽管使用[3H]胸苷掺入法检测T细胞增殖已广泛用于检查DC功能，但该技术仅提供了有关T细胞增殖的有限信息。在这里，我们描述了一种使用流式细胞仪定量分析T细胞增殖的新方法。材料与方法：DCs来自六个健康献血者的CD14 +细胞。使用阳性选择与磁性细胞分选（MACS）分离单核细胞，然后与IL-4，GM-CSF，IL-1beta，IL-6，TNF-α和PGE（2）培养，以产生完全成熟的DC。将HLA I和II类中已知错配的同种异体幼稚T淋巴细胞与DC共培养。没有DC刺激的幼稚T细胞用作阴性对照。在第0、3、5、7、9、11天收获T细胞，并通过流式细胞仪进行分析。 CD3-ECD和CD4-异硫氰酸荧光素（FITC）或CD8-FITC抗体用于区分T细胞亚群，而T细胞活化通过评估HLA-DR，CD45RO，CD25和CD71表达来测量。对于T细胞定量，使用荧光微粒。用7-AAD排除死细胞。还进行了溴脱氧吡啶（BrdU）掺入ELISA程序，以便就绝对细胞计数和CD71表达与T细胞增殖测定进行比较。结果：第1天的初始T细胞浓度为203.9 +/- 39.7（173-265）CD3 + / CD4 +细胞/微升和184.5 +/- 41.6（148-260）CD3 + / CD8 +细胞/微升。在第7天记录了最大的T细胞增殖，T细胞扩增了5到10倍，从而产生了1994.9 +/- 383（1446-2404）CD3 + / CD4 +细胞/微升和944 +/- 303.7（560-1483） CD3 + / CD8 +细胞/微升。此外，两种细胞谱系的活化标记均在第7天（CD71）和第9天（CD25，HLA-DR）上调并达到最大值。 T细胞计数/微升以及CD71表达均与BrdU掺入显着相关。结论：流式细胞仪分析可以简单，精确和快速地定量与DC混合的淋巴细胞反应中的T细胞增殖。激活，增殖和细胞活力可以同时确定。 CD71特别适合用作同时测量T细胞增殖的激活标记。因此，可以详细评估参与抗原特异性增殖的特异性T细胞亚群。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号