Department of Microbiology and Immunology, Baxter Laboratory for Stem Cell Biology, Stanford University School of Medicine, Stanford, California, USA.
Cytometry A. 2010 Nov;77(11):1020-31. doi: 10.1002/cyto.a.20970.
Intracellular flow cytometry permits quantitation of diverse molecular targets at the single-cell level. However, limitations in detection sensitivity inherently restrict the method, sometimes resulting in the inability to measure proteins of very low abundance or to differentiate cells expressing subtly different protein concentrations. To improve these measurements, an enzymatic amplification approach called tyramide signal amplification (TSA) was optimized for assessment of intracellular kinase cascades. First, Pacific Blue, Pacific Orange, and Alexa Fluor 488 tyramide reporters were shown to exhibit low nonspecific binding in permeabilized cells. Next, the effects of antibody concentration, tyramide concentration, and reaction time on assay resolution were characterized. Use of optimized TSA resulted in a 10-fold or greater improvement in measurement resolution of endogenous Erk and Stat cell signaling pathways relative to standard, nonamplified detection. TSA also enhanced assay sensitivity and, in conjunction with fluorescent cell barcoding, improved assay performance according to a metric used to evaluate high-throughput drug screens. TSA was used to profile Stat1 phosphorylation in primary immune system cells, which revealed heterogeneity in various populations, including CD4+ FoxP3+ regulatory T cells. We anticipate the approach will be broadly applicable to intracellular flow cytometry assays with low signal-to-noise ratios.
细胞内流式细胞术允许在单细胞水平上定量测定多种分子靶标。然而，检测灵敏度的限制固有地限制了该方法，有时导致无法测量非常低丰度的蛋白质或区分表达略有不同蛋白质浓度的细胞。为了改善这些测量，优化了一种称为酪胺信号放大（TSA）的酶放大方法，用于评估细胞内激酶级联。首先，显示 Pacific Blue、Pacific Orange 和 Alexa Fluor 488 酪胺报告染料在透化细胞中表现出低非特异性结合。接下来，研究了抗体浓度、酪胺浓度和反应时间对测定分辨率的影响。与标准的非放大检测相比，优化的 TSA 的使用使内源性 Erk 和 Stat 细胞信号通路的测量分辨率提高了 10 倍或更高。TSA 还增强了测定的灵敏度，并且与荧光细胞条形码结合使用，根据用于评估高通量药物筛选的指标提高了测定的性能。TSA 用于分析原发性免疫系统细胞中的 Stat1 磷酸化，这揭示了包括 CD4+FoxP3+调节性 T 细胞在内的各种群体中的异质性。我们预计该方法将广泛适用于信号噪声比较低的细胞内流式细胞术测定。
