高维免疫分析对于研究宿主对免疫疗法的反应至关重要。感染,和鼠类模型系统中的疾病。然而,多参数小组设计的困难加上缺乏现有的鼠类工具,阻碍了在单一试验中对所有主要白细胞表型的全面研究。在这里,我们提出了一个40色流式细胞术小组,用于小鼠淋巴组织的深度免疫表型,包括脾脏,血,Peyer的补丁,腹股沟淋巴结,骨髓,还有胸腺.该小组使用一组强大的表面标记,能够在不使用细胞内染色的情况下区分白细胞亚群,因此,允许在下游功能实验或多组分析中使用细胞。我们的小组对T细胞进行了分类,B细胞,自然杀伤细胞,先天的淋巴样细胞,单核细胞,巨噬细胞,树突状细胞,嗜碱性粒细胞,中性粒细胞,嗜酸性粒细胞,祖细胞,和它们的功能子集通过使用一系列的共刺激,检查点,激活,迁移,和成熟标记。该工具具有多种系统免疫学应用,从循环血液特征的串行监测到复杂的终点分析,特别是在治疗可以调节白细胞丰度和/或功能的临床前设置中。最终,这个40种颜色的面板在时间轴上解析了各种各样的免疫细胞,组织,和治疗,填补了专门用于小鼠免疫分型的现代工具的利基。
High-dimensional immunoprofiling is essential for studying host response to immunotherapy, infection, and disease in murine model systems. However, the difficulty of multiparameter panel design combined with a lack of existing murine tools has prevented the comprehensive study of all major leukocyte phenotypes in a single assay. Herein, we present a 40-color flow cytometry panel for deep immunophenotyping of murine lymphoid tissues, including the spleen, blood, Peyer\'s patches, inguinal lymph nodes, bone marrow, and thymus. This panel uses a robust set of surface markers capable of differentiating leukocyte subsets without the use of intracellular staining, thus allowing for the use of cells in downstream functional experiments or multiomic analyses. Our panel classifies T cells, B cells, natural killer cells, innate lymphoid cells, monocytes, macrophages, dendritic cells, basophils, neutrophils, eosinophils, progenitors, and their functional subsets by using a series of co-stimulatory, checkpoint, activation, migration, and maturation markers. This tool has a multitude of systems immunology applications ranging from serial monitoring of circulating blood signatures to complex endpoint analysis, especially in pre-clinical settings where treatments can modulate leukocyte abundance and/or function. Ultimately, this 40-color panel resolves a diverse array of immune cells on the axes of time, tissue, and treatment, filling the niche for a modern tool dedicated to murine immunophenotyping.