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Phagocytosis of invading pathogens and their subsequent clearance in lysosomes is important for organismal fitness. We have devised the following protocol to extract phagocytic hemocytes from wild-type and mutant Drosophila larvae and infect the isolated hemocytes with GFP-labeled E. coli to measure the rate of phagocytosis and degradation within individual hemocytes over time.
The experiment described below can be used to study phagosome biogenesis, maturation, and delivery to lysosomes. Bacterial accumulation has been well studied in the context of immuno-compromised Drosophila with defects in IMD or Toll signaling and the resulting reduced expression of antimicrobial peptides (e.g., Lemaitre and Hoffmann, 2007; Kleino and Silverman, 2014). Cellular responses to bacterial infections have been less investigated in Drosophila, with most studies focused on mutations that interfere with the initial phagocytic uptake of bacteria by hemocytes (Kocks et al., 2005; Parsons and Foley, 2016). Such bacterial uptake is straightforward to measure using FACS analysis (Tirouvanziam et al., 2004). For a detailed analysis of phagosomal maturation, however, we found it advantageous to examine individual hemocytes attached to a glass cover slip (Akbar et al., 2011; Rahman et al., 2012; Akbar et al., 2016) as this procedure offered us the best combination of temporal and spatial resolution for our studies of phagosome maturation.
To determine the number of remaining bacteria at different time points in different genetic backgrounds, confocal images were opened with ImageJ (NIH) and smoothed with a Gaussian blur of one before merging bacteria and phalloidin-stained channels. The number of bacteria within individual cells was counted and recorded in a Prism (GraphPad) spreadsheet as a single data point for each cell. Bacteria were counted when the entire GFP bacterium was surrounded by phalloidin staining. At least 25 cells were counted for each of three experiments. Prism software was used to plot a box and whisker graph and to perform a one-way ANOVA comparing relevant data sets (all to all).
The authors of this work declare no conflicts of interest.