Drosophila Lamellocyte Origin Figure 8. Proposed model of hemocyte lineage. Current models for the origin of the three hemocyte types propose that plasmatocytes, crystal cells and lamellocytes represent distinct lineages that arise separately from a common stem cell or prohemocyte. We propose that prohemocytes generate either crystal cells or plasmatocytes. Plasmatocytes are a plastic population that can, in turn, generate other hemocyte types such as lamellocytes and podocytes. doi:10.1371/journal.pone.0014051.g008 29uC to inactivate GAL80 and induce Pxn-GAL4-mediated Chn expression. Immunofluorescence microscopy Circulating hemocytes were isolated from five staged wandering third instar larvae and processed for immunofluorescence microscopy using multispot slides (PH-001, C.A.Hendley) as described in [14]. Confocal microscopy was using a Zeiss Axiovert 100 M. Antibodies used were: chicken anti-GFP antibody (1:400, Upstate Biotechnology), mouse MAb L1b (1:60, [31]), mouse MAb H2 anti-Hemese (1:50, [30]), rabbit anti-b-galactosidase (1:2000, Cappel), mouse MAb 40-1a anti-b-galactosidase (1:100), rabbit anti-Histone H3 phospho S10 (H3S10p; 1:2000, Abcam ab5176). Secondary antibodies from Jackson Immunoresearch were: Cy3-conjugated anti-mouse IgG (H+L), FITC-conjugated anti-chicken IgY (H+L), and Cy3-conjugated anti-rabbit IgG (H+L), all used at 1:1000. Chn induction in hemocytes in vitro Virgin w1118; Pxn-GAL4, UAS-GFP; GAL80ts females were crossed with UAS-chn males. Crosses and progeny were maintained at the non-restrictive temperature of 18uC to maintain repression of GAL4. Hemocytes were isolated from 50 wandering stage third instar Pxn-GAL4, UAS-GFP; GAL80ts x UAS-chn larvae that had been cultured at 18uC. Hemocytes were centrifuged at 260 g for 3 minutes at 4uC and resuspended in 500 ml of Schneider’s Drosophila medium (Gibco) containing 10% fetal bovine serum (Invitrogen). 1/5 of the cell suspension was fixed and stained immediately with MAb L1 antibodies. The remaining cell suspension was cultured in 8 well chamber slides (Labtek II) at 29uC for 24 hr. Hemocytes were fixed in 4% paraformaldehyde at room temperature for 10 minutes and stained with MAb L1 antibodies. Marking phagocytically active plasmatocytes Phagocytosis was monitored by injection of fluorescent polystyrene microspheres (Yellow-green 2.0 mm Fluospheres, Invitrogen (F8827)) into the hemocoel of third instar larvae. Fluospheres were vortexed vigorously to ensure even suspension and washed in PBS containing 0.1% BSA (Upstate). Beads were transferred to a drawn glass injection needle (World Precision Instruments, TW100F-4). w1118; Pxn-GAL4, UAS-GFP; GAL80ts x UAS-chn early third instar larvae were immobilized on double- sided tape (Scotch 665, 3 M) attached to a glass slide. Larvae were injected with fluospheres using an Eppendorf Femtojet micro- injector and Leitz Labovert inverted microscope with injection stand and needle holder (Leitz). Injections were made at the posterior of the larvae. Larvae were incubated for 2 hours at room temperature to recover and then transferred to 29uC for 24 hours to induce Chn expression. Hemocytes were prepared for microscopy and stained as described above. Visualizing crystal cells Third instar larval crystal cells were visualized by incubating larvae in 1X PBS for 5 minutes at 60uC. Black crystal cells were observed using an Olympus SZX12 stereo microscope. Cell sorting hopTum-l/+; Pxn-GAL4, UAS-GFP/+ larvae were maintained at 20uC. Hemocytes were isolated from 750 larvae in batches of 50 as described [19]. Hemocytes were centrifuged at 260 g for 3 minutes at 4uC and washed twice in ice cold PBS. Hemocytes were stained with MAb L1 (1:60) for 30 minutes on ice, washed three times in ice cold PBS and stained with biotinylated anti-mouse IgG (H+L) antibodies (Jackson Immunoresearch, 1:1000) for 30 minutes on ice. After three washes with ice cold PBS, hemocytes were stained with APC-streptavidin (BD Pharmingen, 1:750) for 30 minutes on ice, and washes repeated. Cells were filtered through a CellTrics 50 mm filter (Partec) before sorting on a MoFlo high-speed cell sorter (Beckman Coulter). Hemocytes from 100 hopTum-l/+; Pxn- GAL4, UAS-GFP/+ larvae processed as above but not stained with MAb L1 served as a GFP only control. Hemocytes from 100 w1118 larvae processed as above but only stained with biotinylated anti- mouse IgG (H+L) and APC-streptavidin provided a secondary antibody only control to determine baseline APC. Cells were first gated on forward scatter versus side scatter to select viable cells, then on pulse width versus forward scatter to remove doublets, thirdly on compensated FITC signal to select GFP-positive PLoS ONE | www.plosone.org 10 November 2010 | Volume 5 | Issue 11 | e14051