Fluorescence-activated cell sorting (FACS) of Drosophila hemocytes reveals important functional similarities to mammalian leukocytes

Abstract

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 ␤-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.

Functional Evolution of the Vertebrate Myb Gene Family

“…tected by heating the larvae to 70Њ for 10 min, resulting Using a flow cytometry staining strategy that facilitates in the premature melanization of the cells and making the discrimination of Drosophila larval hemocytes from them easily visible through the cuticle of control larvae contaminating events acquired during data collection ( Figure 1A) (Rizki and Rizki 1984). Visible crystal cells (Tirouvanziam et al 2004), we were able to measure were also absent in Dm-Myb, Black Cell double-mutant phagocytosis in living hemocytes. Phagocytosis was larvae, which is of interest because the dominant Black tested by the injection of controlled volumes of fluoresCell mutation causes aberrant melanization of crystal cently labeled, heat-killed E. coli into Dm-Myb Ϫ/Ϫ mosaic cells (data not shown).…”

“…Genes encoding closely related Myb doMyb in the maintenance of genomic integrity and in the suppression of centrosome amplification (Fung et mains have been identified in the cellular slime mold (Dictyostelium discoideum) and in all major plant lineages al. 2002), with ectopic expression of Dm-Myb promoting proliferation in diploid cells and suppressing endore- (Stober-Grasser et al 1992;Braun and Grotewold 1999;Kranz et al 2000). In fact, Myb repeat-containing duplication in endocycling cells (Fitzpatrick et al 2002).…”

DNase II deficiency impairs innate immune function in Drosophila

“…Hemocytes were collected by cutting the larval cuticle with fine forceps under stereomicroscope [31,32]. The number of cells per milliliter was estimated by counting them on a hemocytometer.…”

High-dose oral N -acetylcysteine, a glutathione prodrug, modulates inflammation in cystic fibrosis

“…22. In brief, this method uses the ability of the nonfluorescent probe monochlorobimane to permeate live cells and be conjugated to intracellular GSH by cellular glutathione-S-transferases, thus generating fluorescent GSB adducts (23). Fluorescent adduct formation depends mostly on GSH levels but can also be marginally affected by glutathione-S-transferase activity (which is usually very similar in identical leukocyte subsets from different individuals).…”

“…A direct semiquantitative FACS-based method for measuring neutrophil GSH, which is applicable to unprocessed whole blood samples, was used, coupled with the analytical gating of neutrophils (see Materials and Methods) (22,23). This method was used because neutrophils are very sensitive to external stimulation (19,24).…”