LC3-II is a common marker of autophagosomes in mammalian cells

l culture and preparation B. pseudomallei 1026b, B. pseudomallei DD503, and B. thailandensis E264 were a gift from Don Woods and Glutamax. Neutrophils were incubated at 37uC in 5% CO2 for 20 min before addition of B. pseudomallei or B. thailandensis at an MOI = 1. In specific experiments, diphenyleneiodonium or a DMSO vehicle control was added during this incubation. The plates were centrifuged at 2506g for 5 min to synchronize infection and allowed to incubate at 37uC in 5% CO2 for 10 min before washing 3x with RPMI to remove extracellular bacteria. No antibiotics were used to kill any remaining extracellular bacteria because they have been demonstrated to enter phagocytes and kill intracellular bacteria. The neutrophils in these parallel co-cultures were lysed at either 10 min or 2 h after infection with a 0.5% saponin solution, and intracellular bacteria were enumerated by serial dilution on TSA. Quantification of neutrophil respiratory burst Human neutrophils were seeded at 26105 per well in 96 well plates in PBS containing calcium and magnesium in the presence or absence of DPI. Neutrophils were pretreated with luminol at 37uC in 5% CO2 for 20 min before addition of B. pseudomallei or B. thailandensis at an MOI = 1. Co-cultures were centrifuged at 5006g for 1 min to initiate bacteria:neutrophil contact and immediately analyzed on a FLUOstar Omega plate reader, with luminescence detection every min for 20 min. Statistical analysis Graphpad Instat was used for all statistical analyses. Statistical differences were determined by either performing a two-way T-test or one-way ANOVA followed by a Tukey’s post-hoc test. Results Phagocytosis and clearance of unopsonized Burkholderia 18690793 species by human neutrophils change in bacterial viability of either species after 2 h Indirubin-3′-oxime incubation with neutrophils compared to the initial uptake. Thus, though human neutrophils inherently internalize B. thailandensis more efficiently than B. pseudomallei, they are subsequently unable to clear either bacterial species. Complement deposition on the surface of B. pseudomallei and B. thailandensis Before assessing the effects of serum opsonization on neutrophil responses to B. pseudomallei or B. thailandensis, the relative levels of complement deposition on the surface of these bacteria were measured in the presence of different concentrations of normal human serum, using flow cytometry. After incubation with 5%, 10% and 20% NHS, significant levels of complement component C3 were detected on the surface of both Burkholderia species compared to unopsonized bacteria, whereas 1% NHS opsonization did 10854736 not promote significant C3 deposition. In general, increased serum concentrations correlated with increased C3 deposition in both species. However, B. thailandensis did acquire significantly greater levels of C3 deposition at 5%, 10% and 20% NHS compared to B. pseudomallei. Bacteria incubated with heat-inactivated serum did not acquire substantial C3 on their surfaces. These data indicate that B. Neutrophil Killing of Opsonized B. Pseudomallei deposition at 5% NHS is largely dependent on the classical or lectin pathways and that both are resistant to alternative pathwayactivation at that serum concentration. After incubation in 20% NHS with MgEGTA, C3 binding to B. pseudomallei was significantly reduced compared to in the absence of MgEGTA. This decrease in C3 deposition was not observed for B. thailandensis opsonized in 20% NHS and MgEGTA, demonstrating efficient alterna