Automated analysis of two- and three-color fluorescent Elispot (Fluorospot) assays for cytokine secretion
Automated analysis of two- and three-color fluorescent Elispot (Fluorospot) assays for cytokine secretion
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DOI：
10.1016/j.cmpb.2008.06.002
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发表时间：
2008-10-01
影响因子：
6.1
通讯作者：
Mosmann, Tim R.
中科院分区：
文献类型：
--
作者：
Rebhahn, Jonathan A.;Bishop, Courtney;Mosmann, Tim R.
Elispot 可有效测量分泌特定分子的细胞的频率，尤其是低频细胞，例如抗原特异性 T 细胞。 Fluorospot 测定使这种分析适用于每个细胞两种产物，现在已扩展到小鼠和人类细胞因子分泌细胞的三色测量。由于数据复杂性增加，特别是需要定义单、双和三生产细胞，因此客观量化斑点数量、大小、强度以及与其他斑点的一致性至关重要。因此，开发了一种自动计数程序 Exploraspot，用于检测和量化自动荧光显微镜图像中的荧光点。计算每个点的形态参数，包括大小、强度、位置、圆度等，以 FCS 格式导出，并通过流行的流式细胞术分析程序中的门控和图形显示进行进一步分析。 Exploraspot 的实用性通过单、双和三分泌 T 细胞的鉴定得到证明；可变背景荧光的耐受性；以及真实和随机多重事件数量的估计。 (C) 2008 Elsevier Ireland Ltd. 保留所有权利。
The Elispot effectively measures the frequencies of cells secreting particular molecules, especially low-frequency cells such as antigen-specific T cells. The Fluorospot assay adapted this analysis to two products per cell, and this has now been extended to three-color measurement of both mouse and human cytokine-secreting cells. Due to the increased data complexity, and particularly the need to define single-, double- and triple-producing cells, it is critical to objectively quantify spot number, size, intensity, and coincidence with other spots. An automated counting program, Exploraspot, was therefore developed to detect and quantify Fluorospots in automated fluorescence microscope images. Morphological parameters, including size, intensity, location, circularity and others are calculated for each spot, exported in FCS format, and further analyzed by gating and graphical display in popular flow cytometry analysis programs. The utility of Exploraspot is demonstrated by identification of single-, double- and triple-secreting T cells; tolerance of variable background fluorescence; and estimation of the numbers of genuine versus random multiple events. (C) 2008 Elsevier Ireland Ltd. All rights reserved.