Garvan Institute of Medical Research, Darlinghurst, NSW, Australia.
School of Clinical Medicine, Sydney, NSW, Australia.
Methods Mol Biol. 2024;2826:189-199. doi: 10.1007/978-1-0716-3950-4_14.
The use of flow cytometry for immunophenotyping is contingent on the ability to accurately assign biological relevance to the detected signal. This process has historically been challenging when defining IgE expressing B cells or IgE expressing antibody-secreting cells due to widespread expression of receptors for IgE on various leukocyte subsets, including human B cells. Here we describe our implementation of intracellular staining for human IgE following a blocking step to negate the challenge of surface-bound IgE. We also describe our experience with a human B cell culture system that can be used to robustly validate this approach before application to primary human samples. Orthogonal confirmatory techniques remain essential; these are not described in detail, but several possible strategies are suggested.
流式细胞术用于免疫表型分析的前提是能够准确地将检测到的信号赋予生物学意义。在定义表达 IgE 的 B 细胞或表达 IgE 的抗体分泌细胞时，由于 IgE 受体在各种白细胞亚群（包括人类 B 细胞）中广泛表达，因此这一过程在历史上一直具有挑战性。在这里，我们描述了在阻断步骤之后进行人 IgE 的细胞内染色的实施，以消除表面结合 IgE 的挑战。我们还描述了我们在人 B 细胞培养系统方面的经验，该系统可用于在将该方法应用于原代人样本之前对其进行稳健验证。正交确认技术仍然是必不可少的；这些技术没有详细描述，但提出了几种可能的策略。
