Laboratory of Dermatology and Immunodeficiencies (LIM56), Tropical Medicine Institute (IMT), School of Medicine, São Paulo University, São Paulo, Brazil.
Methods Mol Biol. 2025;2857:15-31. doi: 10.1007/978-1-0716-4128-6_3.
B cells are crucial components of the immune system, responsible for producing specific antibodies in response to infections and vaccines. Despite their uniform appearance, B cells display diverse surface molecules and functional properties, which are not yet fully understood. Apart from antibody production, B cells also play roles in antigen presentation and cytokine secretion, essential for initiating T-cell immune responses. Their significance as disease biomarkers and therapeutic targets has led to increased research focus. However, the lack of standardized protocols for B-cell identification and the variability in defining B-lymphocyte subpopulations pose some challenges. This paper proposes a B-cell identification panel throughout the evaluation of previous cytometry panels and nomenclature heterogeneity for B-cell subpopulations. Major subpopulations recognized in human peripheral blood include transitional, naive, switched memory, unswitched memory, double negative, and plasmablasts, characterized based on their functional and phenotypic features. We present a standardized flow cytometry protocol utilizing surface phenotypic markers (CD3, CD19, IgD, CD27, CD38, and CD24) to differentiate and analyze B-cell subpopulations. This practical and cost-effective panel can be used in various research and laboratory settings. The challenges of standardizing names and markers for classifying B-lymphocyte subpopulations are discussed, along with protocols utilizing multiple markers and gating strategies, allied with the importance of considering viability markers. In summary, this standardized protocol and panel provide a comprehensive approach to identifying B-cell subpopulations to enhance the reproducibility and comparability of B-cell subpopulation studies.
B 细胞是免疫系统的重要组成部分，负责产生针对感染和疫苗的特异性抗体。尽管 B 细胞外观均一，但它们表现出多样化的表面分子和功能特性，这些特性尚未完全了解。除了抗体产生外，B 细胞还在抗原呈递和细胞因子分泌中发挥作用，对于启动 T 细胞免疫反应至关重要。它们作为疾病生物标志物和治疗靶点的重要性导致了对其研究的增加。然而，缺乏用于 B 细胞鉴定的标准化方案以及 B 淋巴细胞亚群定义的可变性仍然是一些挑战。本文提出了一个贯穿于以前的细胞术面板评估和 B 细胞亚群命名异质性的 B 细胞鉴定面板。在人类外周血中识别的主要亚群包括过渡性、幼稚性、开关记忆性、未开关记忆性、双阴性和浆母细胞，其特征基于其功能和表型特征。我们提出了一个标准化的流式细胞术方案，利用表面表型标志物（CD3、CD19、IgD、CD27、CD38 和 CD24）来区分和分析 B 细胞亚群。这个实用且具有成本效益的面板可用于各种研究和实验室设置。本文还讨论了标准化 B 淋巴细胞亚群分类的名称和标志物的挑战，以及利用多个标志物和门控策略的方案，同时强调了考虑活力标志物的重要性。总之，这个标准化的方案和面板提供了一种全面的方法来识别 B 细胞亚群，以提高 B 细胞亚群研究的可重复性和可比性。
