Laboratory of Functional Genomics, Institute of Genetics, Biological Research Centre, Szeged H-6726, Hungary; University of Szeged, Ph.D. School in Biology, Szeged H-6726, Hungary.
Immunology Unit, Institute of Genetics, Biological Research Centre, Szeged H-6726, Hungary.
Genomics Proteomics Bioinformatics. 2021 Apr;19(2):243-252. doi: 10.1016/j.gpb.2020.06.022. Epub 2021 Mar 10.
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, weoptimizedSCMC 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) and carrying a deficiency of the tumor suppressor gene lethal(3)malignant blood neoplasm-1 [l(3)mbn], as well as in stem cell maintenance-defective hdc mutant larvae. Multidimensional analysis enabled the discrimination of the functionally different major hemocyte subsets for lamellocytes, plasmatocytes, and crystal cells, anddelineated 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)mbn and hop, 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.
单细胞质量细胞术（SCMC）结合了传统流式细胞术（即荧光激活细胞分选）和质谱的特点，使得在单细胞水平上对生物调节机制进行复杂分析成为可能。在这项研究中，我们优化了 SCMC 来分析果蝇先天免疫系统的血细胞。我们使用金属偶联的抗体（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，同时使用抗 GFP 来检测免疫诱导样本中的晶体细胞。我们研究了在未诱导状态、免疫诱导状态、携带果蝇同源 Janus 激酶（hop）驱动突变和携带肿瘤抑制基因致死（3）恶性血液肿瘤-1 [l(3)mbn]缺陷的肿瘤突变体以及在干细胞维持缺陷 hdc 突变幼虫中的单个细胞和血细胞群体的抗原表达谱。多维分析能够区分功能不同的主要血细胞亚群，如扁平血细胞、浆血细胞和晶体细胞，并描绘了果蝇突变体的独特免疫表型。我们在肿瘤株 l(3)mbn 和 hop 中分别鉴定出了 L2/P1 和 L2/L4/P1 过渡表型细胞的亚群，以及免疫诱导后的 L4/P1 细胞亚群。我们的结果首次表明，SCMC 结合多维生物信息学分析，代表了一种强大的工具，可以深入分析果蝇细胞介导免疫的调节。
