An excitation wavelength of 480 nm and an emission wavelength of 520 nm were utilized

e points. These data demonstrate that autophagy is induced in mast cells following P. aeruginosa infection, and exposure to the bacteria promotes flux 26225771 through the pathway. We further characterized the dose response of autophagy induced by P. aeruginosa strain 8821, PAK and PAO.1 as well as E. coli stain DH5a. A dose-dependent increase in LC3-GFP puncta formation in HMC-1 5C6 cells was observed in response to all strains of bacteria suggesting that the induction of autophagy in mast cells may be a generalized host response to P. aeruginosa and other gram negative bacteria. As previous reports have suggested that LC3 becomes incorporated into the membranes of mast cell granules, we set out to ensure the LC3 Vesnarinone chemical information positive structures we observed were not mast cell granules. Because the fixation conditions required for staining mast cell granules are acidic, the HMC-1 5C6 LC3-GFP cell line could not be used to examine colocalization of LC3 positive puncta with mast cell granules as GFP has a pKa of 6.0 and is not fluorescent under acidic conditions. In order to visualize LC3 localization under these conditions HMC-1 5C6 cells were transiently transfected with LC3-mCherry, which retains fluorescence under acidic pH. LC3-mCherry expressing cells were then fixed and stained for mast cell granules using toluidine blue and subjected to fluorescent and light microscopy. Consistent with previous reports, the HMC-1 5C6 cells were not well granulated, with only 21% of cells containing granules. Of the granulated cells there was on average only 7 granules per cell. The number of mast cell granules and the number of LC3-mCherry positive mast cell granules were counted in 100 cells containing at least one granule, and one LC3-mCherry positive puncta. Colocalization between LC3 and mast cell granules was very rarely observed. Furthermore, there was no correlation between the number of LC3-mCherry positive puncta, and the number of mast cell granules in a given cell. Together these results indicate that LC3 positive structures in HMC-1 5C6 cells are not mast cell granules. P. aeruginosa induces autophagy in primary human and mouse mast cells and becomes incorporated into autophagosomes We next set out to examine the ultrastructural characteristics of autophagosomes in primary human and murine mast cells. To address this question we infected primary human cord blood derived mast cells and primary mouse BMMCs with P. aeruginosa strain 8821 at an MOI of 1:100. Eight hours later cells were fixed and processed for TEM viewing. Highly vesicularized double membrane bound vesicles characteristic of autophagosomes were observed in both untreated and P. aeruginosa treated cells. The percentage of cytosol contained within autophagosomes, as well as the number of autophagosomes per cross-section was significantly increased 3630970 in P. aeruginosa treated cells. Furthermore, P. aeruginosa bacteria were repeatedly seen inside the cell contained within double membrane bound vesicles, although these observations were infrequent. Together these results demonstrate that autophagy is induced by P. aeruginosa in primary human and mouse mast cells, and that the bacteria can become incorporated into autophagosomes in vitro. Autophagy contributes to bacterial killing by mast cells in vitro following P. aeruginosa infection Having observed bacteria inside autophagosome like structures, we next set out to examine the impact of autophagy on bacterial killing in mast cells following P. aerugin