Roederer M, De Rosa S, Gerstein R, Anderson M, Bigos M, Stovel R, Nozaki T, Parks D, Herzenberg L, Herzenberg L
Department of Genetics, Stanford University, California, USA.
Cytometry. 1997 Dec 1;29(4):328-39. doi: 10.1002/(sici)1097-0320(19971201)29:4<328::aid-cyto10>3.0.co;2-w.
We developed the chemistry, instrumentation, and software technologies needed to measure, simultaneously and independently, eight different fluorescent molecules on individual cells. Conjugation of these fluorochromes to monoclonal antibodies is straightforward; all immunofluorescence staining is accomplished with direct stains only. We built a hybrid flow cytometer with eight fluorescence detectors and two light scatter channels, with excitation provided by three spatially separated laser beams emitting at 407 nm, 488 nm, and 595 nm. The fluorescence compensation required to make the data orthogonal is of sufficient complexity that it cannot be performed manually; thus, we use software to compensate the data post hoc, based on data collected from singly stained compensation control samples. In this report, we evaluate the 8 color staining technology. Of the seven fluorochromes other than fluorescein, six have a useful brightness at least as great as fluorescein. Three of the fluorochromes (phycoerythrin, allophycocyanin, and the Cy5 resonance energy tandem of phycoerythrin) are considerably brighter than fluorescein and are useful for detecting antigens expressed at low levels. Finally, we show the power and utility of the 8 color, 10-parameter technology using staining experiments on both human and murine immune systems.
我们开发了在单个细胞上同时独立测量八种不同荧光分子所需的化学、仪器和软件技术。将这些荧光染料与单克隆抗体结合很简单；所有免疫荧光染色仅通过直接染色完成。我们构建了一台具有八个荧光探测器和两个光散射通道的混合流式细胞仪，由三个空间分离的激光束提供激发光，分别发射407nm、488nm和595nm的光。使数据正交所需的荧光补偿非常复杂，无法手动完成；因此，我们使用软件根据从单染色补偿对照样本收集的数据进行事后数据补偿。在本报告中，我们评估了八色染色技术。除荧光素外的七种荧光染料中，有六种具有至少与荧光素一样高的有用亮度。其中三种荧光染料（藻红蛋白、别藻蓝蛋白和藻红蛋白的Cy5共振能量串联体）比荧光素亮得多，可用于检测低水平表达的抗原。最后，我们通过对人类和小鼠免疫系统进行染色实验，展示了八色十参数技术的强大功能和实用性。
