hemocytes, and finally on compensated APC to select MAb L1- negative cells. Gates were set to select GFP-positive MAb L1- negative hemocytes. Hemocytes were sorted into Schneider’s Drosophila medium (Gibco, 11720) containing 10% fetal bovine serum (Invitrogen) and cultured in 8 well chamber slides (Labtek II) at 29uC for 30 hours. Hemocytes were subsequently fixed with 4% paraformaldehyde at room temperature for 10 minutes and stained with MAb L1 antibodies and processed for confocal microscopy as described above. Wasp infestation Lineage tracing experiments were performed by crossing virgin females of the genotype y 1 , w1118, UAS-Flp.Exel; act5C.stop.lacZ with w1118; Pxn-GAL4, UAS-GFP males and the progeny main- tained at 25uC. Late second instar/early third instar larvae were infested with 5 females of Leptopilina boulardi G486 at 25uC for 12 hours. Hemocytes were prepared from larvae aged for 30 hours and stained using MAb L1 and anti-lacZ antibodies. RNAi-mediated knockdown of Chn was achieved by crossing w1118, P{UAS-Dcr-2.D}1; Pxn-GAL4, UAS-GFP virgin females with UAS-chni line 35507 provided by the Vienna Drosophila RNAi Center. Control (driver crossed with w1118 males) and knockdown 2nd instar larvae were infested with L. boulardi as described above, aged for 30 hours and lamellocytes visualized using MAb L1. Real-time PCR analysis of transcript levels Hemocytes were isolated from a total of 300 third instar larvae of each genotype in batches of fifty animals. Larvae were bled into HyQ CCM3 culture media containing protease inhibitors and hemocytes pelleted by centrifugation at 260 g for 5 minutes at 4uC. Hemocytes were washed twice with PBS. mRNA was isolated from hemocytes using a mMACS mRNA Isolation kit (Miltenyi Biotec Ltd, UK) according to the manufacturers instructions. For analysis of transcript abundance in third instar larvae, mRNA was isolated from 5 w1118 larvae using mMACS mRNA Isolation kit. cDNA was generated by reverse transcription using Superscript II (Invitrogen) at 42uC. Real-time PCR was performed on an Applied Biosystems Step One Plus real-time PCR machine. Reactions were performed using Absolute QPCR SYBR green ROX mix (Thermo Fisher Scientific, AB-1162). Primers used are described in Table S1. RpL32 transcript provided the endogenous control for normalization. Supporting Information Figure S1 Chn over-expression doe not affect crystal cell development. (A) Pxn-GAL4, UAS-GFP x w1118 and (B) Pxn- GAL4, UAS-GFP x UAS-chn third instar larvae were heat-treated to reveal crystal cells (black cells in right panels). Chn over-expression disrupts crystal cell accumulation at sessile compartments but does not decrease crystal cell number. Found at: doi:10.1371/journal.pone.0014051.s001 (4.36 MB TIF) Figure S2 Chn induced-lamellocyte differentiation is cell autonomous. Hemocytes isolated from (A) Hml-GAL4, UAS-GFP/ UAS-chn and (B) He-GAL4, UAS-GFP/UAS-chn third instar larvae References Drosophila Lamellocyte Origin were stained with MAb L1 (red) and anti-GFP (green) antibodies. Nuclei were also stained using DAPI (shown in purple). Hemocytes that are both GFP- and MAb L1-positive could be detected (closed arrowheads). GFP-positive hemocytes that do not stain with MAb L1 are indicated (open arrowheads). Scalebar represents 50 mm. (C) Lamellocyte frequencies observed after Chn expression using Pxn-GAL4 and Hml-GAL4 were normalized to the percentage of hemocytes that express GAL4 in the Pxn-GAL4 and Hml-GAL4 driver lines. The resultant ratio (transdetermination frequency) shows that Pxn-GAL4 and Hml-GAL4 have similar abilities to induce lamellocyte differentiation. Found at: doi:10.1371/journal.pone.0014051.s002 (0.92 MB TIF) Figure S3 Control of Pxn-GAL4 mediated expression by GAL80ts. (A,B) Schematic illustrating the TARGET system. (A) A ubiquitously expressed, temperature-sensitive GAL80 variant (GAL80ts) is functional at 18uC. GAL80ts binds to and represses GAL4 expressed by Pxn-GAL4 in plasmatocytes, to repress GAL4- mediated transcription of responders like UAS-GFP. (B) At 29uC, GAL80ts is inactivated and GAL4 expressed by Pxn-GAL4 in plasmatocytes can activate transcription of targets like UAS-GFP. (C-H) (GAL80ts) can regulate Pxn-GAL4 mediated gene expression in hemocytes. Hemocytes were isolated from (C) Pxn-GAL4, UAS- GFP third instar larvae, (D) Pxn-GAL4, UAS-GFP; GAL80ts third instar larvae raised 18uC, and (E) Pxn-GAL4, UAS-GFP; GAL80ts third instar larvae raised at 18uC but shifted to 29uC for 24 hr. Hemocytes were stained with anti-GFP antibodies (green) and DAPI (blue) to reveal nuclei. GFP is detected in Pxn-GAL4, UAS- GFP hemocytes, and Pxn-GAL4, UAS-GFP; GAL80ts hemocytes at 29uC, but is not detected in hemocytes from Pxn-GAL4, UAS-GFP; GAL80ts larvae raised at 18uC. GFP expression in (C) and (E) is equivalent. (F–H) Live intact larvae of the equivalent genotypes to (C–E) showing GFP fluorescence in dorsal sessile hemocyte compartments (dashed ellipse). (F) GFP-expressing hemocytes are detected in sessile compartments in Pxn-GAL4, UAS-GFP larvae. (G) The sessile patches are indistinguishable from the surrounding tissues at 18uC but (H) become clearly visible after 24 hr temperature upshift at 29uC. Found at: doi:10.1371/journal.pone.0014051.s003 (4.40 MB TIF) Table S1 Primers used for real-time PCR analysis. Found at: doi:10.1371/journal.pone.0014051.s004 (0.10 DOC) MB Acknowledgments We thank I. Ando, M. Galko, D. Hultmark, H. Ja¨ckle, J. Kumar, M. Williams and the Bloomington Drosophila Stock Center for generously providing flies and reagents. We thank C. Buckley, B.Y. Jang, M. Mortin and M. Watson for critical reading of the manuscript; and M. Krajcovic and M.A. Kim for preparing fly-food. Author Contributions Conceived and designed the experiments: MS SYK PB. Performed the experiments: MS SYK PB. Analyzed the data: MS SYK PB. Wrote the paper: SYK PB. 1. Meister M, Lagueux M (2003) Drosophila blood cells. Cell Microbiol 5: 573–580. 2. Hartenstein V (2006) Blood cells and blood cell development in the animal kingdom. Annu Rev Cell Dev Biol 22: 677–712. 3. Lemaitre B, Hoffmann J (2007) The host defense of Drosophila melanogaster. Annu Rev Immunol 25: 697–743. 4. Jones SL, Lindberg FP, Brown EJ (1999) Phagocytosis. Philadelphia: Lippincott- Raven Publishers. 5. Soderhall K, Cerenius L (1998) Role of the prophenoloxidase-activating system in invertebrate immunity. Curr Opin Immunol 10: 23–27. 6. Rizki TM, Rizki RM (1992) Lamellocyte differentiation in Drosophila larvae parasitized by Leptopilina. Dev Comp Immunol 16: 103–110. 7. Sorrentino RP, Carton Y, Govind S (2002) Cellular immune response to parasite infection in the Drosophila lymph gland is developmentally regulated. Dev Biol 243: 65–80. PLoS ONE | www.plosone.org 11 November 2010 | Volume 5 | Issue 11 | e14051