Macrophage-Cryptococcus neoformans Assay

The macrophage-C. neoformans co-incubation assay was adapted from [23 (link)]. J774A.1 macrophage-like murine cells grown in DMEM (Dulbecco’s Modified Eagle Medium, Sigma-Aldrich) were harvested, washed in PBS, transferred to 96-well tissue culture plates at 10⁵ cells/well, and incubated overnight at 37 °C and 5% CO₂. The C. neoformans strain H99 was incubated in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) overnight with shaking at 37 °C, washed with PBS, and resuspended in DMEM medium at 10³ cells/ml. Sphaerostilbellins (1 and 2) were prepared in serial 2-fold dilutions in DMEM and DMEM containing H99 cells to achieve a dose range of 0.03–32 µM. The macrophage medium was replaced with the DMEM medium containing compound or fungal cells plus compound and incubated for 24 to 48 h at 37 °C and 5% CO₂. Macrophage viability was assessed by the addition of 10% alamarBlue and incubating for 3 h at 37 °C and 5% CO₂ prior to fluorescence measurement (FLUORStar Optima plate reader, BMG Labtech, Cary, NC, USA). Fungal growth was assessed visually at 48 h. To test for fungal survival, samples from each well were plated onto YPD agar and incubated at 30 °C. Each compound was tested in duplicate biological replicates.