采用全光谱流式细胞术分析原代鼠 T 细胞中 T 细胞受体诱导的钙离子内流。
Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry.
机构信息
Department of Immunology and Microbiology, University of Colorado-Anschutz School of Medicine.
Department of Immunology and Microbiology, University of Colorado-Anschutz School of Medicine;
出版信息
J Vis Exp. 2022 Dec 16(190). doi: 10.3791/64526.
Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to assess calcium signaling by band-pass flow cytometry. This protocol is designed to measure calcium responses in primary murine T-cells using full spectrum flow cytometry. Total splenocytes are labeled with the ratiometric calcium indicator dye Indo-1, along with a panel of fluorochrome-conjugated antibodies to cell surface molecules. Leveraging the capabilities of full spectrum flow cytometry provides a platform for utilizing a wide array of cell surface stains in combination with Indo-1. Cells are then analyzed in real-time at 37 °C before and after the addition of an anti-CD3 antibody to stimulate the T-cell receptor. After unmixing the spectral signals, the ratio of calcium-bound to calcium-free Indo-1 is calculated and can be visualized over time for each gated population of splenocytes. This technique can allow for the simultaneous analysis of calcium responses in multiple cell populations.
钙离子内流是 T 细胞受体刺激的常见反应，也是 T 细胞信号转导的常用测量方法。已经开发出几种钙指示剂染料，通过带通流式细胞术来评估钙信号转导。本方案旨在使用全光谱流式细胞术测量原代小鼠 T 细胞的钙反应。用比率型钙指示剂 Indo-1 以及一组荧光染料标记的细胞表面分子抗体对总脾细胞进行标记。充分利用全光谱流式细胞术的功能，为利用 indo-1 与多种细胞表面染色相结合提供了一个平台。然后，在添加抗 CD3 抗体刺激 T 细胞受体之前和之后，在 37°C 下实时分析细胞。在解混光谱信号后，计算钙结合与游离 Indo-1 的比率，并可以随时间为每个门控脾细胞群体可视化。该技术可实现多个细胞群体钙反应的同时分析。
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