Quantifying mCherry E. coli in Drosophila Hemocytes

To quantify the number of mCherry E. coli bacteria (K12 E. coli expressing PDSpRSETD-cherry plasmid from Dr. Stephan Mesnage) in hemocytes in vivo, He-Gal4; UAS-FYVE-GFP or he-Gal4; UAS-FYVE-GFP, hemo^A4 flies were injected with 0.2 μL of bacterial culture grown to OD600 = 0.05, using a glass capillary microinjection needle. Flies were then dissected as described in Magny et. al 2013 [7 (link)] to expose the dorsal abdominal cuticle in order to image the hemocytes associated to the dorsal vessel, as described in Horn et al. 2014 [71 (link)]. Flies were dissected after 20 or 120 min postinjection, in PBS, and the cuticles fixed in 4% PFA in PBS for 20 min. The preparations were then washed in, PBTX, incubated 30 min in PBS with phalloidin-Cy5 (Sigma-Aldrich) (1:10), washed in PBS, and mounted in Vectashield (vector). Hemocytes were imaged with a Zeiss laser scanning microscope LSM 5.10 on a Zeiss Axioskop 2 stage, with a 40X Achroplan objective, and bacterial cells within hemocytes, as determined with the phalloidin and FYVE counter-stains, were counted in Z-stack reconstructions.