Single-cell mass cytometry (SCMC) combines features of traditional flow cytometry (i.e., fluorescence-activated cell sorting) with mass spectrometry, making it possible to measure several parameters at the single-cell level for a complex analysis of biological regulatory mechanisms. In this study, we optimized SCMC to analyze hemocytes of the Drosophila innate immune system. We used metal-conjugated antibodies (against cell surface antigens H2, H3, H18, L1, L4, and P1, and intracellular antigens 3A5 and L2) and anti-IgM (against cell surface antigen L6) to detect the levels of antigens, while anti-GFP was used to detect crystal cells in the immune-induced samples. We investigated the antigen expression profile of single cells and hemocyte populations in naive states, in immune-induced states, in tumorous mutants bearing a driver mutation in the Drosophila homologue of Janus kinase (hopTum) and carrying a deficiency of the tumor suppressor gene lethal(3)malignant blood neoplasm-1 [l(3)mbn1], as well as in stem cell maintenance-defective hdcΔ84 mutant larvae. Multidimensional analysis enabled the discrimination of the functionally different major hemocyte subsets for lamellocytes, plasmatocytes, and crystal cells, and delineated the unique immunophenotype of Drosophila mutants. We have identified subpopulations of L2+/P1+ and L2+/L4+/P1+ transitional phenotype cells in the tumorous strains l(3)mbn1 and hopTum, respectively, and a subpopulation of L4+/P1+ cells upon immune induction. Our results demonstrated for the first time that SCMC, combined with multidimensional bioinformatic analysis, represents a versatile and powerful tool to deeply analyze the regulation of cell-mediated immunity of Drosophila.
单细胞质谱（SCMC）结合了传统流式细胞术（FACS）和质谱的功能，可以在单细胞水平上测量多个参数，以进行复杂的生物学调节机制分析。在这项研究中，我们优化了SCMC以分析果蝇先天免疫系统的血细胞。我们使用金属结合抗体（在细胞表面的H2，H3，H18，L1，L4和P1，在细胞内的3A5和L2）和免疫球蛋白M 抗体（在细胞表面的L6）来检测抗原水平，而绿色荧光蛋白抗体（anti-GFP）用于检测免疫诱导样品中的含晶细胞。我们研究了未诱导状态下，和免疫诱导状态下单细胞和血细胞群体的抗原表达谱。我们对携带果蝇Janus激酶（hopTum）的果蝇驱动突变并携带缺乏肿瘤抑制因子l(3)mbn1基因的肿瘤突变体中进行了调查，并对在干细胞维持缺陷型hdcΔ84突变体幼虫中也进行了调查。多维分析能够区分功能不同的主要血细胞亚群：薄层细胞，浆细胞和含晶细胞，并描绘了果蝇突变体的独特免疫表型。我们已经发现了肿瘤菌株中L2 +/P1 +(l(3)mbn1), L2+/L4+/P1+ (hopTum) 过渡表型细胞的亚群以及免疫诱导后的L4+/P1+细胞的亚群。我们的结果首次证明，单细胞质谱与多维生物信息学分析相结合，代表了一种多功能且功能强大的工具，可深入分析果蝇细胞介导的免疫调节。





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