Lysotracker red dnd 99 solution

To quantify circulating hemocytes, L3 wandering larvae were dissected on 12-spot microscope slides (Hendley-Essex, Essex, UK) in Drosophila Ringer’s solution containing 0.01% 1-phenyl-2-thiourea (Sigma Aldrich). Hemocytes were left to adhere for 45 min, and then fixed with acetone for 6 min. Samples were mounted with PBS-glycerol and were analyzed with a Zeiss Axioskope 2 MOT microscope (Carl Zeiss) using phase contrast mode. For phagocytosis experiments, L3 wandering larvae were washed in Drosophila Ringer’s solution, and placed on a dry paper towel. Larvae were injected with FITC-labeled E. coli using a glass capillary. After 1 hr, larvae were dissected on 12-spot microscope slides in Drosophila Ringer’s solution containing 0.01% 1-phenyl-2-thiourea. Hemocytes were left to adhere for 45 min, then lysosomes were stained with 0.1 µM LysoTracker Red DND-99 solution (Thermo Fisher) for 5 min. Surface-bound bacteria were quenched with 0.4% trypan blue solution, and samples were analyzed on an Olympus FLUOVIEW FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan).

For visualization of Lc3 dynamics, Tg(CMV:EGFP-map1lc3b) larvae were embedded in 1.5% low-melting-point agarose (weight per volume, in egg water) and imaged using a Leica TCS SPE confocal microscope. Imaging was performed using a ×63 oil immersion objective (HC PL APO CS2, numerical aperture [NA] of 1.42) in a region of the tail fin to detect enhanced GFP (EGFP)-map1lc3b (GFP-Lc3)-positive vesicles. For quantification of acidic vesicles in the presence and absence of infection, larvae were immersed in embryo medium containing 5 μM LysoTracker Red DND-99 solution (Thermo Fisher Scientific) for 1 h. Before mounting and imaging, larvae were washed three times with embryo medium. To determine colocalization between Mm and GFP-Lc3 or LysoTracker, fixed (GFP-Lc3) or live anesthetized (LysoTracker) larvae were embedded in 1.5% low-melting-point agarose (in egg water) and imaged in the caudal hematopoietic tissue using a Leica TCS SP8 confocal microscope with a ×40 water immersion objective (HCX APO L U-V-I, NA of 0.8). Images were obtained using Leica Las X software. For the quantification of GFP-Lc3 levels, the find maxima algorithm with a noise tolerance of 50 was used in Fiji software version 1.53c. To determine association of GFP-Lc3 or LysoTracker with bacteria, manual counting was performed on the obtained confocal images using Leica Las X software.