Wang Lili, Gaigalas Adolfas K, Abbasi Fatima, Marti Gerald E, Vogt Robert F, Schwartz Abe
National Institute of Standards and Technology, Gaithersburg, MD 20899-8312.
Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, NIH Building 29B, Room 2NN08, Bethesda, MD 20892.
J Res Natl Inst Stand Technol. 2002 Aug 1;107(4):339-53. doi: 10.6028/jres.107.027. Print 2002 Jul-Aug.
The present work uses fluorescein as the model fluorophore and points out critical steps in the use of MESF (Molecules of Equivalent Soluble Fluorophores) values for quantitative flow cytometric measurements. It has been found that emission spectrum matching between a reference solution and an analyte and normalization by the corresponding extinction coefficient are required for quantifying fluorescence signals using flow cytometers. Because of the use of fluorescein, the pH value of the medium is also critical for accurate MESF assignments. Given that the emission spectrum shapes of microbead suspensions and stained biological cells are not significantly different, the percentage of error due to spectrum mismatch is estimated. We have also found that the emission spectrum of a microbead with a seven-methylene linker between the fluorescein and the bead surface (bead7) provides the best match with the spectra from biological cells. Therefore, bead7 is potentially a better calibration standard for flow cytometers than the existing one that is commercially available and used in the present study.
本研究以荧光素作为模型荧光团，并指出了在定量流式细胞术测量中使用MESF（等效可溶性荧光团分子）值的关键步骤。研究发现，使用流式细胞仪对荧光信号进行定量时，需要参考溶液与分析物之间的发射光谱匹配，并通过相应的消光系数进行归一化。由于使用了荧光素，介质的pH值对于准确的MESF赋值也至关重要。鉴于微珠悬浮液和染色生物细胞的发射光谱形状没有显著差异，估计了由于光谱不匹配导致的误差百分比。我们还发现，在荧光素与微珠表面之间具有七个亚甲基连接基团的微珠（bead7）的发射光谱与生物细胞的光谱匹配最佳。因此，与本研究中使用的市售现有校准标准相比，bead7可能是流式细胞仪更好的校准标准。
