Faculty of Dentistry, University of Toronto, Toronto, ON, Canada.
Department of Dental Oncology, Princess Margaret Cancer Centre, Toronto, Canada.
Curr Protoc. 2024 Mar;4(3):e1020. doi: 10.1002/cpz1.1020.
Flow cytometry stands as the most employed high-throughput single-cell analysis technique, facilitating the profiling of remarkably diverse samples, such as blood, bone marrow and body fluids. In addition, it allows for the discrimination of diverse immune cell subsets, including infrequently encountered types like T regulatory cells and exhausted CD28 T cells. However, analyzing rare immune cell subsets with conventional flow cytometry poses challenges stemming from factors like fluorophore overlap, compensation issues, and limited flexibility in fluorophore selection. Therefore, spectral flow cytometry offers advantages over traditional flow cytometry. It measures the full emission spectrum and then separates it to identify different fluorochromes. This enables the use of fluorochromes with significant overlap in a single test, allowing for the analysis of more protein markers. Following this, spectral technology employs precise calculations to separate individual fluorochromes, thereby enabling the detection and elimination of autofluorescent signals originating from cells within the entire emission spectrum. This capability is pivotal in achieving deep phenotyping of immune cells with the requisite sensitivity and resolution essential for monitoring the immune systems of patients with compromised immunity, such as cancer and autoimmune disorders. Additionally, it allows for the exploration of interactions between distinct immune subsets. In this context, we introduce an optimized protocol utilizing spectral flow cytometry for precise T-cell characterization and differentiation, encompassing the assessment of their activation states. Furthermore, this protocol extends its applicability to the identification of less common circulating T-cell populations, notably T-regulatory and CD28 T cells, following autofluorescence correction within the spectrum. This protocol provides a set of steps and reagents for the surface and intracellular staining of human T cells using whole peripheral blood. The spectral-based design of this panel allows for its applicability to other spectral machines, providing a versatile and efficient tool for T-cell analysis. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Achieving optimal staining through effective antibody titration Basic Protocol 2: Single-cell staining Basic Protocol 3: Comprehensive panel staining post-titration and spectral library integration.
流式细胞术是目前应用最广泛的高通量单细胞分析技术，可用于分析多种类型的样本，如血液、骨髓和体液。此外，它还可以区分不同的免疫细胞亚群，包括罕见的调节性 T 细胞和耗尽型 CD28 T 细胞。然而，使用传统流式细胞术分析罕见的免疫细胞亚群存在一些挑战，如荧光染料重叠、补偿问题以及在荧光染料选择方面的局限性。因此，光谱流式细胞术相对于传统流式细胞术具有优势。它测量整个发射光谱，然后将其分离以识别不同的荧光染料。这使得可以在单个测试中使用具有显著重叠的荧光染料，从而能够分析更多的蛋白质标志物。接下来，光谱技术使用精确的计算来分离单个荧光染料，从而可以检测和消除来自整个发射光谱中细胞的自发荧光信号。这种能力对于实现对免疫细胞的深度表型分析至关重要，这对于监测免疫功能受损患者的免疫系统（如癌症和自身免疫性疾病患者）的敏感性和分辨率至关重要。此外，它还可以用于探索不同免疫亚群之间的相互作用。在此，我们介绍了一种利用光谱流式细胞术进行精确 T 细胞特征分析和分化的优化方案，包括评估其激活状态。此外，该方案还通过在光谱内进行自发荧光校正，扩展了其对罕见循环 T 细胞群体（如 T 调节细胞和 CD28 T 细胞）的识别能力。该方案提供了一组使用全外周血对人 T 细胞进行表面和细胞内染色的步骤和试剂。该面板的基于光谱的设计使其可适用于其他光谱仪器，为 T 细胞分析提供了一种多功能、高效的工具。© 2024 Wiley Periodicals LLC. 基本方案 1：通过有效的抗体滴定实现最佳染色 基本方案 2：单细胞染色 基本方案 3：全面面板染色后的滴定和光谱库整合。
