Phagocytosis and killing assays in BMDMs and PMNs

For the killing assays, 10⁶ BMDMs were left to adhere in 6-well plates for 1 h in 2 ml of DMEM complete media at 37 °C in a 5% CO₂ incubator, and subsequently infected with either R. oryzae or A. fumigatus conidia, at an MOI of 1:1. BMDMs were washed three times with warm PBS 30 min after the infection to remove non-phagocytosed conidia. At the indicated time point of infection (2 or 6 h), BMDMs were harvested by scraping, placed in Eppendorf tubes, lysed by sonication (three times for 10 s and once for 5 s for A. fumigatus-infected and R. oryzae-infected BMDMs, respectively), centrifuged at 1000 rpm for 10 min at 4 °C, and the pellet containing intracellular conidia was resuspended in 200 μl sterile PBS. Aspergillus fumigatus killing was assessed as previously described using propidium iodide staining²². For the evaluation of killing of Rhizopus conidia by BMDMs, intracellular conidia recovered after BMDM lysis were incubated at 37 °C in a 5% CO₂ incubator with DMEM complete medium for ~4 h, until germination was microscopically observed. Killing of R. oryzae was assessed using a Bürker counting chamber based on the percentage of germinating conidia. Germination of intracellular Rhizopus conidia by BMDMs was always normalized to the germination of control R. oryzae conidia following sonication (5 s) and cultured in DMEM complete medium for ~4 h in the absence of BMDMs, which was typically always >95%. In representative experiments, killing of A. fumigatus was also assessed based on the germination assay.

For phagocytosis assay, BMDMs and polymorphonuclear neutrophils (PMNs) from GFP-LC3 mice were stimulated with R. oryzae and A. fumigatus conidia at an MOI of 2:1 at 37 °C in a 5% CO₂ incubator for different time points. Cells were then fixed and stained for confocal microscopy as previously mentioned. Phagocytic index was expressed with the following formula: (total number of engulfed cells/total number of counted macrophages) × (number of macrophages containing engulfed cells/total number of counted macrophages) × 100.