Lovelace Patricia, Maecker Holden T
Human Immune Monitoring Center, Institute for Immunity, Transplantation, and Infection, Stanford University, Fairchild Science Building, 299 Campus Drive, Stanford, CA, 94305-5124, USA.
Methods Mol Biol. 2018;1678:151-166. doi: 10.1007/978-1-4939-7346-0_9.
Intracellular cytokine staining is a popular method for visualizing cellular responses, most often T-cell responses to antigenic or mitogenic stimulation. It can be coupled with staining for other functional markers, such as upregulation of CD107 or CD154, as well as phenotypic markers that define specific cellular subsets, e.g., effector and memory T-cell compartments, NK cells, or monocytes. Recent advances in multicolor flow cytometry instrumentation and software have allowed the routine combination of 12 or more markers, creating some technical and analytical challenges along the way, and exposing a need for standardization in the field. Here, we will review best practices for antibody panel design and procedural variables for multicolor intracellular cytokine staining, and present an optimized protocol with variations designed for use with specific markers and sample types.
细胞内细胞因子染色是一种用于可视化细胞反应的常用方法，最常用于检测T细胞对抗原或有丝分裂原刺激的反应。它可以与其他功能标志物的染色相结合，如CD107或CD154的上调，以及定义特定细胞亚群的表型标志物，例如效应和记忆T细胞亚群、自然杀伤细胞或单核细胞。多色流式细胞术仪器和软件的最新进展使得能够常规组合12种或更多标志物，在此过程中带来了一些技术和分析挑战，并凸显了该领域标准化的必要性。在这里，我们将回顾抗体组合设计的最佳实践以及多色细胞内细胞因子染色的程序变量，并提出一种优化方案，该方案针对特定标志物和样本类型进行了变体设计。
