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 (hop^Tum) 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 hdc^Δ84 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 hop^Tum, 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.

单细胞质量流式细胞术(SCMC) 将传统流式细胞术（即荧光激活细胞分选）的特征与质谱法相结合，可以在单细胞水平上测量多个参数，从而对生物调控机制进行复杂分析。在这项研究中，我们 优化 了 SCMC 以分析果蝇的血细胞先天免疫系统。我们使用金属结合抗体（针对细胞表面抗原 H2、H3、H18、L1、L4 和 P1，以及细胞内抗原 3A5 和 L2）和抗 IgM（针对细胞表面抗原 L6）来检测抗原水平，而抗GFP用于检测免疫诱导样品中的晶体细胞。我们研究了单细胞和血细胞群在幼稚状态、免疫诱导状态、带有Janus 激酶 ( hop ^Tum ) 的果蝇同系物驱动突变且携带致死的肿瘤抑制基因缺陷的肿瘤突变体的抗原表达谱(3)malignant blood neoplasm-1 [ l(3)mbn ¹ ]，以及干细胞维持缺陷型hdc^Δ84突变幼虫。多维分析能够区分板层细胞、浆细胞和晶体细胞的功能不同的主要血细胞亚群，并 描绘了果蝇突变体的独特免疫表型。我们分别在肿瘤菌株l(3)mbn ¹和hop ^{Tum中鉴定了 L2 +} /P1 ⁺和 L2 ⁺ /L4 ⁺ /P1 ⁺过渡表型细胞亚群，以及 L4 ⁺ /P1 ^+亚群免疫诱导后的细胞。我们的研究结果首次证明，SCMC 与多维生物信息学分析相结合，代表了深入分析果蝇细胞介导免疫调节的多功能且强大的工具。