Laboratory for Functional Immune Repertoire Analysis, Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zürich.
Laboratory for Functional Immune Repertoire Analysis, Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zürich; Department of Biomedicine, Aarhus University;
J Vis Exp. 2024 Mar 8(205). doi: 10.3791/66492.
Infections, autoimmune diseases, desired and adverse immunological responses to treatment can lead to a complex and dynamic cytokine response in vivo. This response involves numerous immune cells secreting various cytokines to orchestrate the immune reaction. However, the secretion dynamics, amounts, and co-occurrence of the different cytokines by various cell subtypes remain poorly understood due to a lack of appropriate tools to study them. Here, we describe a protocol using a microfluidic droplet platform that allows the time-resolved quantitative measurement of secretion dynamics for several cytokines in parallel on the single-cell level. This is enabled by the encapsulation of individual cells into microfluidic droplets together with a multiplexed immunoassay for parallel quantification of cytokine concentrations, their immobilization for dynamic fluorescent imaging, and the analysis of the respective images to derive secreted quantities and dynamics. The protocol describes the preparation of functionalized magnetic nanoparticles, calibration experiments, cell preparation, and the encapsulation of the cells and nanoparticles into droplets for fluorescent imaging and subsequent image and data analysis using the example of lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The presented platform identified distinct cytokine secretion behavior for single and co-secreting cells, characterizing the expected phenotypic heterogeneity in the measured cell sample. Furthermore, the modular nature of the assay allows its adaptation and application to study a variety of proteins, cytokines, and cell samples, potentially leading to a deeper understanding of the interplay between different immune cell types and the role of the different cytokines secreted dynamically to shape the tightly regulated immune response. These new insights could be particularly interesting in the studies of immune dysregulations or in identifying target populations in therapy and drug development.
感染、自身免疫性疾病、对治疗的期望和不良反应免疫应答会导致体内复杂而动态的细胞因子反应。这种反应涉及到许多免疫细胞分泌各种细胞因子来协调免疫反应。然而，由于缺乏适当的研究工具，各种细胞亚型分泌的不同细胞因子的分泌动力学、数量和共同发生仍然知之甚少。在这里，我们描述了一种使用微流控液滴平台的方案，该方案允许在单细胞水平上并行实时定量测量几种细胞因子的分泌动力学。这是通过将单个细胞封装在微流控液滴中，以及同时进行细胞因子浓度的多重免疫分析、细胞因子的固定化以进行动态荧光成像、以及对各自图像的分析来实现的，从而可以推导出分泌量和动力学。该方案描述了功能化磁性纳米粒子的制备、校准实验、细胞制备以及将细胞和纳米粒子封装到液滴中用于荧光成像，以及使用脂多糖刺激的人外周血单核细胞的实例进行后续图像和数据分析。所提出的平台确定了单个和共分泌细胞的不同细胞因子分泌行为，从而表征了所测量的细胞样本中预期的表型异质性。此外，该测定的模块化性质允许对其进行适应和应用，以研究各种蛋白质、细胞因子和细胞样本，从而可能更深入地了解不同免疫细胞类型之间的相互作用以及动态分泌的不同细胞因子在塑造紧密调节的免疫反应中的作用。这些新的见解在免疫失调的研究或在治疗和药物开发中确定目标人群方面可能特别有趣。
