Aleith Johann, Bergmann-Ewert Wendy, Müller-Hilke Brigitte
Core Facility for Cell Sorting and Cell Analysis, Rostock University Medical Center, Rostock, Germany.
Eur J Immunol. 2025 Mar;55(3):e202451193. doi: 10.1002/eji.202451193.
Intracellular cytokine labeling combined with high-parametric flow cytometry offers substantial promise in elucidating the nuanced effector functions of cells. However, the establishment of complex multicolor panels is often laborious and the importance of validation processes may be underestimated in research practice. This raises the risk of prematurely translating multicolor panels into in vivo studies. Alternatively, researchers may resort to animal disease models to procure cytokine-producing cells. Both scenarios raise ethical concerns as they entail the potential for unnecessary animal suffering without yielding novel insights into immunobiology. Here, we perform multicolor panel optimization and validation without the need for stressful animal testing. We designed two spectral flow cytometry panels for cytokine expression analyses across mouse immune and joint cells. Animal testing was replaced by stimulated co-cultures of T cells, splenocytes, and fibroblast-like synoviocytes. These cultures were used for multicolor labeling experiments. Our method proved suitable for validating the two cytometry panels, as it provided a complex cellular environment in which a variety of cytokine-producing populations were identified. In summary, we here present a blueprint for the quality control of single-cell cytokine assays by cell culture and further introduce multicolor panels that can be employed for studies on inflammatory or infectious diseases.
细胞内细胞因子标记与高参数流式细胞术相结合，在阐明细胞细微的效应器功能方面具有巨大潜力。然而，复杂多色面板的建立往往很费力，而且在研究实践中，验证过程的重要性可能被低估。这增加了过早将多色面板应用于体内研究的风险。另外，研究人员可能会借助动物疾病模型来获取产生细胞因子的细胞。这两种情况都引发了伦理问题，因为它们都有可能导致动物不必要的痛苦，却无法对免疫生物学产生新的见解。在此，我们无需进行压力较大的动物试验即可进行多色面板的优化和验证。我们设计了两个光谱流式细胞术面板，用于分析小鼠免疫细胞和关节细胞中的细胞因子表达。用T细胞、脾细胞和成纤维样滑膜细胞的刺激共培养物替代动物试验。这些培养物用于多色标记实验。我们的方法被证明适用于验证这两个细胞术面板，因为它提供了一个复杂的细胞环境，在其中可以识别出多种产生细胞因子的细胞群体。总之，我们在此展示了通过细胞培养进行单细胞细胞因子检测质量控制的蓝图，并进一步介绍了可用于炎症或传染病研究的多色面板。
