A flow cytometric method to estimate the precursor frequencies of cells proliferating in response to specific antigens
A flow cytometric method to estimate the precursor frequencies of cells proliferating in response to specific antigens
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DOI：
10.1016/s0022-1759(99)00136-2
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发表时间：
1999-11-19
影响因子：
2.2
通讯作者：
Wallace, PK
中科院分区：
文献类型：
--
作者：
Givan, AL;Fisher, JL;Wallace, PK
荧光染料对细胞膜或细胞质进行染色，然后在子细胞分裂时进行分裂，与流式细胞术一起使用来测量细胞的增殖。在这篇文章中，我们利用外周血单个核细胞对破伤风环状病毒的反应，描述了这种染色方法的扩展，以计算抗原特异性T细胞的前体频率。利用荧光直方图的数学去卷积提供关于每个子代中的细胞比例的信息，可以导出关于响应特定刺激的原始种群中的细胞的前体频率的信息，来自具有不同比例的固定和存活细胞的模型系统的数据表明，流动方法返回准确的前体频率值。根据流式细胞仪数据采集的特点，估计NOW方法可以检测到在添加刺激之前代表大约1/10(5)人口的细胞的增殖。当将结果与限制稀释技术的结果进行比较时，流式细胞仪方法返回的值表明前体频率较高。讨论了造成这种差异的可能原因。流式细胞术方法具有简单的优点，同时还具有对反应细胞进行表型鉴定和确定其增殖率的额外能力。Flow方法可作为过敏、移植排斥和自身免疫领域的一种简单、常规的分析方法，并可用于量化疫苗接种和癌症免疫治疗的反应。(C)1999 Elsevier Science B.V.保留所有权利。
Fluorescent dyes that stain cell membranes or cytoplasm and then partition between daughter cells at division have been used in conjunction with flow cytometry to measure the proliferation of cells. In this paper, using peripheral blood mononuclear cells responding to tetanus toroid, we describe an extension of this dye methodology to calculate the precursor frequency of antigen-specific T-cells. With mathematical deconvolution of the fluorescence histograms providing information about the proportion of cells in each of the daughter generations, information can be derived about the precursor frequency of cells in the original population that responded to the specific stimulus, Data from a model system with different proportions of fixed and viable cells indicate that the flow method returns accurate values for precursor frequency. Based on the characteristics of flow cytometric data acquisition, it is estimated that the now method could detect proliferation of cells that represented, before addition of the stimulus, approximately 1/10(5) of the population. When comparing results to those from the limiting dilution technique, the flow cytometric method returns values that indicate higher precursor frequencies. Possible reasons for this discrepancy are discussed. The flow cytometric method offers the advantage of simplicity as well as the additional ability to phenotype the responding cells and determine their rate of proliferation. The flow method may find use as a simple, routine assay in the fields of allergy, transplant rejection, and autoimmunity and for quantitating responses to vaccination and cancer immunotherapy. (C) 1999 Elsevier Science B.V. All rights reserved.