Freer Giulia
Department of Translational Medicine, Retrovirus Center, University of Pisa, Via del Brennero 2, I-56127, Pisa, Italy,
Methods Mol Biol. 2014;1172:221-34. doi: 10.1007/978-1-4939-0928-5_20.
The detection of cytokines inside cells producing them has made a tremendous impact on the way immune reactivity is measured. Intracellular cytokine staining is the only immunological technique allowing determination of antigen-specific T cell function and phenotype at the same time; for this reason, it is one of the most popular methods to measure antigenicity in the evaluation of vaccine efficacy and in the study of infectious diseases. It is a flow cytometric technique based on staining of intracellular cytokines and cell markers (surface or cytoplasmic) with fluorescent antibodies after short term culture of stimulated immune cells in the presence of a protein secretion inhibitor, followed by fixation and permeabilization. Most experiments involve detection of five to ten different colors but many more can be detected by modern flow cytometers. Here, we discuss our experience using a standard protocol for intracellular cytokine staining.
对产生细胞因子的细胞内细胞因子进行检测，极大地影响了免疫反应性的测量方式。细胞内细胞因子染色是唯一一种能够同时测定抗原特异性T细胞功能和表型的免疫学技术；因此，它是评估疫苗效力和研究传染病时测量抗原性最常用的方法之一。这是一种流式细胞术技术，其原理是在存在蛋白质分泌抑制剂的情况下对刺激后的免疫细胞进行短期培养，然后用荧光抗体对细胞内细胞因子和细胞标志物（表面或细胞质）进行染色，随后进行固定和通透处理。大多数实验涉及检测五到十种不同颜色，但现代流式细胞仪可以检测更多颜色。在此，我们讨论使用细胞内细胞因子染色标准方案的经验。
