Tirouvanziam Rabindra, Davidson Colin J, Lipsick Joseph S, Herzenberg Leonard A
Herzenberg Laboratory, Department of Genetics, Stanford University School of Medicine, Beckman Center B007, Stanford, CA 94305-5318, USA.
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):2912-7. doi: 10.1073/pnas.0308734101. Epub 2004 Feb 19.
Drosophila is a powerful model for molecular studies of hematopoiesis and innate immunity. However, its use for functional cellular studies remains hampered by the lack of single-cell assays for hemocytes (blood cells). Here we introduce a generic method combining fluorescence-activated cell sorting and nonantibody probes that enables the selective gating of live Drosophila hemocytes from the lymph glands (larval hematopoietic organ) or hemolymph (blood equivalent). Gated live hemocytes are analyzed and sorted at will based on precise quantitation of fluorescence levels originating from metabolic indicators, lectins, reporters (GFP and beta-galactosidase) and antibodies. With this approach, we discriminate and sort plasmatocytes, the major hemocyte subset, from lamellocytes, an activated subset present in gain-of-function mutants of the Janus kinase and Toll pathways. We also illustrate how important, evolutionarily conserved, blood-cell-regulatory molecules, such as calcium and glutathione, can be studied functionally within hemocytes. Finally, we report an in vivo transfer of sorted live hemocytes and their successful reanalysis on retrieval from single hosts. This generic and versatile fluorescence-activated cell sorting approach for hemocyte detection, analysis, and sorting, which is efficient down to one animal, should critically enhance in vivo and ex vivo hemocyte studies in Drosophila and other species, notably mosquitoes.
果蝇是造血和先天免疫分子研究的强大模型。然而，由于缺乏血细胞（血细胞）的单细胞检测方法，其在功能性细胞研究中的应用仍然受到限制。在此，我们介绍一种结合荧光激活细胞分选和非抗体探针的通用方法，该方法能够从淋巴腺（幼虫造血器官）或血淋巴（相当于血液）中选择性地分选活的果蝇血细胞。基于对源自代谢指标、凝集素、报告基因（绿色荧光蛋白和β-半乳糖苷酶）和抗体的荧光水平的精确量化，对分选的活血细胞进行随意分析和分选。通过这种方法，我们从类浆细胞（一种在Janus激酶和Toll途径功能获得性突变体中存在的活化亚群）中区分并分选了主要的血细胞亚群——浆细胞。我们还说明了如何在血细胞内对钙和谷胱甘肽等重要的、进化上保守的血细胞调节分子进行功能研究。最后，我们报告了分选的活血细胞的体内转移以及从单个宿主中检索后对其成功进行的重新分析。这种用于血细胞检测、分析和分选的通用且多功能的荧光激活细胞分选方法，即使在低至一只动物的情况下也很有效，应该会显著增强果蝇和其他物种（特别是蚊子）体内和体外的血细胞研究。
