Drosophila Lamellocyte Origin Figure 4. Endogenous Chn expression. (A) chn transcript levels are elevated in hemocyte populations relative to whole larvae. Elevation of chn transcript levels parallels that observed for the hemocyte expressed He and Hml transcripts. In contrast chn transcripts are reduced in fat body relative to whole larvae. Data are mean and standard deviation of three independent determinations (B) RNAi knockdown of Chn reduces lamellocyte differentiation observed after wasp infestation. Lamellocyte frequencies were determined after MAb L1 staining. Data are mean and standard deviation of 5 confocal panels from each of 4 separate infested larvae hemocyte preparations. doi:10.1371/journal.pone.0014051.g004 Figure 5. In vitro differentiation of lamellocytes from plasmatocytes. (A–D) hopTum-l/+; Pxn-GAL4-UAS-GFP/+ larvae raised at 20uC contain hemocytes intermediate between mature lamellocytes and plasmatocytes. (A) MAb L1 and (C) anti-GFP staining. (B) Merge showing overlap in MAb L1 (red) and anti-GFP (green) Small (,20 mm) GFP+/MAb L12 plasmatocytes (closed arrows), large (.40 mm) GFP2/MAb L1+ lamellocytes (closed arrowheads) and intermediate sized (,40 mm) GFP+/MAb L1+ hemocytes (open arrowheads) were detected. (D) 24.7% of hemocytes are the intermediate GFP+/MAb L1+ population. No large (.40 mm) GFP+/MAb L1+ lamellocytes were detected. Data are mean +/2 s.d. of 8 determinations. (E–I) FACS purified GFP+/MAb L12 plasmatocytes from hopTum-l/+; Pxn-GAL4-UAS-GFP/+ larvae can be induced to differentiate into lamellocytes in vitro at 29uC. (E) Hemocytes before sorting, stained with MAb L1 (red) and anti-GFP antibodies (green) Populations are labeled as in (B). (F) Hemocytes were FACS sorted to select GFP+/MAb L12 cells (lower right quadrant) (G) Confocal microscopy confirms sorted cells as GFP+/MAb L12 plasmatocytes. (H) These can differentiate into MAb L1+ lamellocytes after in vitro culture at 29uC for 30 hr. Both large mature lamellocytes (closed arrowheads) and (,40 mm) intermediates (open arrowheads) were detected. (I) Increase in lamellocyte frequency after culture at 29uC (30 hr) Data are the mean +/2 s.d. of three independent determinations. In (E,G,H) DAPI-stained nuclei are shown in purple. Scalebar in all panels denotes 50 mm. doi:10.1371/journal.pone.0014051.g005 PLoS ONE | www.plosone.org 7 November 2010 | Volume 5 | Issue 11 | e14051