Krutzik Peter O, Clutter Matthew R, Trejo Angelica, Nolan Garry P
Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, California.
Curr Protoc Cytom. 2011 Jan;Chapter 6:6.31.1-6.31.15. doi: 10.1002/0471142956.cy0631s55.
Fluorescent cell barcoding (FCB) enables high throughput, high content flow cytometry by multiplexing samples prior to staining and acquisition on the cytometer. Individual cell samples are barcoded, or labeled, with unique signatures of fluorescent dyes so that they can be mixed together, stained, and analyzed as a single sample. By mixing samples prior to staining, antibody consumption is typically reduced 10- to 100-fold. In addition, data robustness is increased through the combination of control and treated samples, which minimizes pipetting error, staining variation, and the need for normalization. Finally, speed of acquisition is enhanced, enabling large profiling experiments to be run with standard cytometer hardware. In this unit, we outline the steps necessary to apply the FCB method to cell lines, as well as primary peripheral blood samples. Important technical considerations, such as choice of barcoding dyes, concentrations, labeling buffers, compensation, and software analysis, are discussed.
荧光细胞条形码技术（FCB）通过在细胞仪上进行染色和采集之前对样品进行多重化处理，实现了高通量、高内涵流式细胞术。单个细胞样品用荧光染料的独特标记进行条形码编码或标记，以便它们可以混合在一起，进行染色，并作为单个样品进行分析。通过在染色前混合样品，抗体消耗通常可减少10至100倍。此外，通过将对照样品和处理样品相结合，数据稳健性得以提高，这最大限度地减少了移液误差、染色差异以及归一化的需求。最后，采集速度得到提高，使得大型分析实验能够使用标准细胞仪硬件进行。在本单元中，我们概述了将FCB方法应用于细胞系以及原代外周血样品所需的步骤。还讨论了重要的技术考虑因素，如条形码染料的选择、浓度、标记缓冲液、补偿和软件分析。
