Approaches to Quantitation in Flow Cytometry
Approaches to Quantitation in Flow Cytometry
复制标题
流式细胞术定量方法
DOI：
--
复制
发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Lili Wang
中科院分区：
文献类型：
--
作者：
A. Gaigalas;Lili Wang
流式细胞术用于测量T细胞和B细胞表面抗原标记的荧光强度（FI）。T细胞和B细胞与人类适应性免疫系统有关，表面抗原的数量，如CD4、CD8和CD20，可用于诊断目的。为了估计表面上表达的特定抗原的数量，将细胞孵育在含有该抗原特异性抗体的溶液中，抗体与荧光团结合，荧光团提供荧光信号，用于检测细胞表面上抗体的存在。抗体、微球或细胞上的荧光团称为标签。该抗体具有生物特异性，荧光团提供了一种读出机制。通过将标记细胞的荧光信号与标记的参考微球的荧光信号进行比较来完成细胞仪测量的定量。来自标记细胞和标记微球的荧光信号被转换为荧光产率（FY），其中FY定义为荧光团数量和荧光团量子产率的乘积。荧光的比较为估计与细胞表面上的标记相关的等效可溶性荧光团（MESF）的分子数量提供了基础。概述了将MESF值分配给微球的程序，然后演示了将MESF值分配给固定化r -藻红蛋白微球。MESF值用于获得与细胞结合的抗体（ABCe）的估计。定量过程的最后一步是将ABCe值转换为细胞表面上特定抗原数量的估计。如果注意适当，ABCe值可以很好地指示细胞表面存在的特定抗原的总数。给出了测定淋巴细胞上CD4和CD20抗原数量的例子。MESF定量策略已应用于多色流式细胞仪的标定。
Flow cytometry is used to measure the fluorescence intensity (FI) from labels bound to antigens present on the surface of T and B cells. The T and B cells are associated with the human adaptive immune system and the amounts of surface antigens, such as CD4, CD8, and CD20, are used for diagnostic purposes. To estimate the number of a specific antigen expressed on the surface, the cells are incubated in a solution containing antibodies specific to that antigen and the antibodies are conjugated to fluorophores that provide the fluorescence signals used to detect the presence of the antibodies on the cell surface. The fluorophore on the antibody, microsphere, or cell is called a label. The antibody gives biological specificity and the fluorophore provides a mechanism for readout. Quantitation of cytometer measurements is accomplished by the comparison of the fluorescence signal of labeled cells with the fluorescence signal of labeled reference microspheres. The fluorescence signals from labeled cells and labeled microspheres are converted to a fluorescence yield (FY) where FY is defined as the product of the number of fluorophores and the fluorophore quantum yield. The comparison of fluorescence provides the basis for an estimate of the number of molecules of equivalent soluble fluorophores (MESF) associated with the labels bound on the cell surface. A procedure is outlined for assigning MESF values to microspheres followed by a demonstration of the assignment of MESF values to microspheres with immobilized R-phycoerythrin. The MESF values are used to obtain an estimate of the antibodies bound to the cell (ABCe). The final step in the quantitation process is the conversion of the ABCe values to an estimate of the number of specific antigens on a surface of a cell. With proper care, the ABCe value may be a good indicator of the total number of specific antigens present on the cell surface. Examples are given of the determination of the number of CD4 and CD20 antigens on lymphocytes. The MESF quantitation strategy has been applied to the calibration of multicolor flow cytometers.
影响因子：
2.9
作者：
Huang, SC;Stump, MD;Caldwell, KD
通讯作者：
Caldwell, KD