Oonishi T, Uyesaka N
J Immunol Methods. 1985 Nov 28;84(1-2):143-54. doi: 10.1016/0022-1759(85)90423-5.
A standard fluorescence microsphere has been prepared for quantitative flow cytometry. A uniformly sized (6.53 +/- 0.34 micron in diameter) and perfectly spherical plastic microsphere, Micropearl-FT, was covalently conjugated with fluorescein isothiocyanate (FITC). The characteristic of this microsphere to lyse completely in strong alkaline solutions made it possible to determine the amount of bound FITC easily and reproducibly. The amount of bound FITC depended on the concentration of FITC and the incubation time at pH 9.5 and 37 degrees C. In flow cytometry, the fluorescence intensity of the FITC-conjugated microsphere (FCMS) showed linear dependence on the amount of bound FITC. The fluorescence intensity of FCMS was stable for at least 3 months even in aqueous solutions. Using FCMS, an Epics V was calibrated and the minimum measurable fluorescence intensity was examined. From analysis using this system, the number of FITC-conjugated OKT3 molecules bound to human T lymphocytes was shown to average 4.4 X 10(4) per cell at saturation.
已制备出一种用于定量流式细胞术的标准荧光微球。一种尺寸均匀（直径为6.53±0.34微米）且完美呈球形的塑料微球Micropearl-FT与异硫氰酸荧光素（FITC）共价结合。这种微球在强碱性溶液中能完全裂解的特性使得能够轻松且可重复地测定结合的FITC量。结合的FITC量取决于FITC的浓度以及在pH 9.5和37℃下的孵育时间。在流式细胞术中，FITC偶联微球（FCMS）的荧光强度与结合的FITC量呈线性相关。即使在水溶液中，FCMS的荧光强度至少3个月内保持稳定。使用FCMS对Epics V进行了校准，并检测了最小可测荧光强度。通过该系统的分析表明，在饱和状态下，与人类T淋巴细胞结合的FITC偶联OKT3分子数量平均为每个细胞4.4×10⁴个。
