Krutzik Peter O, Nolan Garry P
Department of Microbiology and Immunology, Baxter Laboratory in Genetic Pharmacology, Stanford University, Stanford, California 94305, USA.
Nat Methods. 2006 May;3(5):361-8. doi: 10.1038/nmeth872.
Flow cytometry allows high-content, multiparameter analysis of single cells, making it a promising tool for drug discovery and profiling of intracellular signaling. To add high-throughput capacity to flow cytometry, we developed a cell-based multiplexing technique called fluorescent cell barcoding (FCB). In FCB, each sample is labeled with a different signature, or barcode, of fluorescence intensity and emission wavelengths, and mixed with other samples before antibody staining and analysis by flow cytometry. Using three FCB fluorophores, we were able to barcode and combine entire 96-well plates, reducing antibody consumption 100-fold and acquisition time to 5-15 min per plate. Using FCB and phospho-specific flow cytometry, we screened a small-molecule library for inhibitors of T cell-receptor and cytokine signaling, simultaneously determining compound efficacy and selectivity. We also analyzed IFN-gamma signaling in multiple cell types from primary mouse splenocytes, revealing differences in sensitivity and kinetics between B cells, CD4+ and CD4- T cells and CD11b-hi cells.
流式细胞术能够对单细胞进行高内涵、多参数分析，使其成为药物发现和细胞内信号通路分析的一种有前景的工具。为了给流式细胞术增加高通量能力，我们开发了一种基于细胞的多重分析技术，称为荧光细胞条形码（FCB）。在FCB中，每个样品都用不同的荧光强度和发射波长的特征标记或条形码进行标记，并在抗体染色和流式细胞术分析之前与其他样品混合。使用三种FCB荧光团，我们能够对整个96孔板进行条形码标记和合并，将抗体消耗减少100倍，每板采集时间缩短至5 - 15分钟。使用FCB和磷酸化特异性流式细胞术，我们筛选了一个小分子文库以寻找T细胞受体和细胞因子信号通路的抑制剂，同时确定化合物的功效和选择性。我们还分析了来自原代小鼠脾细胞的多种细胞类型中的γ干扰素信号通路，揭示了B细胞、CD4 +和CD4 - T细胞以及CD11b高表达细胞之间在敏感性和动力学方面的差异。
