5.6. Macrophage Lysis Assay

Bone marrow-derived macrophages (BMDM) from 8 week-old female C57/Bl6 mice (a gift from Hooke Laboratories, Lawrence, MA, USA) were isolated from marrow differentiated in the presence of macrophage colony stimulating factor (CSF−1) as previously described [66]. Macrophage infections and lactate dehydrogenase (LDH) release assays were carried out using BMDM media [Dulbecco’s modified eagle medium (DMEM) without phenol red (Gibco-ThermoFisher, Waltham, MA, USA) supplemented with 20% fetal bovine serum (Cytiva HyClone, Marlborough, MA, USA) 2 mM glutamine, 1 mM Na-pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin, 400 μg/mL uracil and 20 ng/mL M-CSF (Gibco-ThermoFisher, Waltham, MA, USA). Phenol red was omitted from the media due to interference with the detection wavelength used for the LDH release assay. BMDM were seeded at a density of 2 × 10⁵ cells/well in 24-well tissue culture treated dishes (Corning- CoStar, Cambridge, MA, USA) and allowed to adhere for two hours. Separately, log phase H. capsulatum WU15 yeast cultures were pelleted, resuspended in DMEM without phenol red and counted by hemacytometer. Yeasts were added to macrophage wells at a multiplicity of infection (MOI) of 5. After a 2 h incubation period to allow for yeasts to be phagocytosed, the media was removed and macrophage monolayers were washed twice with DMEM without phenol red. Following macrophage washing, 750 µL BMDM media was added to each well. The infected macrophages were incubated at 37 °C with 5% CO_2. At 24 h post-infection, 250 µL fresh media was added to each well. At 48 h following infection, LDH levels in BMDM supernatants were measured using the CytoTox Non-Radioactive cytotoxicity kit (Promega, Madison, WI, USA) as previously described [44,66]. The % lysis at each time point was calculated relative to LDH levels measured in uninfected BMDM artificially induced to lyse using 1% Triton X−100. Three independent experiments were performed.