Imager 72

Tsetse flies (Glossina morsitans) were maintained at 27°C and 70% hygrometry in Roubaud cages, in groups of 50 male flies per cage. Pleomorphic trypanosome cell lines were maintained at 1 × 10⁵ cells/ml density in HMI-9 medium plus 10% FBS at 37°C with 5% CO₂. In vitro stumpy differentiation was induced in HMI-9, supplemented with 10% FBS without antibiotics, by adding 8-pCPT-2′-O-Me-5′-AMP (5 μM) (BioLog-Life Science Institut) to the culture 48 h before fly infection (Laxman et al., 2006 (link)). On the day of infection, trypanosomes were resuspended at 10⁶ cells per ml in SDM79 with no antibiotics supplemented with 10 mM glutathione prior infection (MacLeod et al., 2007 (link)). Flies were fed on infected media through a silicone membrane and maintained until dissection by feeding three times per week on sheep’s blood in heparin. Flies were starved for 2 days before dissection at day 28. Imaging was carried out using a ZEISS Imager 72 epifluorescence microscope with an Axiocam 506 mono camera. Single images (for DIC) or multichannel stacks (for fluorescence) of images every 0.24 µm were acquired. When maximum intensity Z-projections are presented, they were generated using Fiji (Schlindelin et al., 2012 (link)).

Immunofluorescence analysis was carried out using standard protocols as described previously [37 (link)]. Rabbit anti-VSG2 was used at 1:20 000 and rabbit anti -γH2A [41 (link)] was used at 1:250. Fluorescein-conjugated goat α-rabbit and goat anti-mouse secondary antibodies (Pierce) were used at 1:2000. Samples were mounted in Vectashield (Vector Laboratories) containing 4, 6-diamidino-2-phenylindole (DAPI). In T. brucei, DAPI-stained nuclear and mitochondrial DNA can be used as cytological markers for cell cycle stage [42 (link)]; one nucleus and one kinetoplast (1N:1K) indicate G₁, one nucleus and an elongated kinetoplast (1N:eK) indicate S phase, one nucleus and two kinetoplasts (1N:2K) indicate G₂/M and two nuclei and two kinetoplasts (2N:2K) indicate post-mitosis. Images were captured using a ZEISS Imager 72 epifluorescence microscope with an Axiocam 506 mono camera and images were processed in ImageJ. For both γH2A and G2 counts, 2 independent inductions were performed and all counts were done by 2 individual researchers