Fix permeabilization buffer

Pulmonary macrophages were isolated as described above. Macrophages were then incubated with C. neoformans cna1Δ at a 1:1 ratio at 37°C in 5% CO₂ in RPMI without phenol red plus 10% heat-inactivated fetal bovine serum (Life Technologies) for 2 h. Golgi plug (Brefeldin A; BD Biosciences) was added according to manufacturer's recommendations and incubated for an additional 4 h. (6 h. total). Cells were washed with 1X PBS and stained with Zombie viability dye (Cat No 423104; Biolegend) at room temperature in the dark for 15 mins then washed with 1x PBS. Cells were stained for surface markers CD45, CD64, and CD11b (S3 Table) and incubated at 4°C for 30 mins. Cells were washed and fixed with Fix/Permeabilization buffer (BD Biosciences) for 20 mins. Cells were washed with 1X Perm/wash buffer (BD Biosciences) and stained for intracellular markers IFN-γ, IL-2, and TNF-α (S3 Table) for 30 min at 4°C. Cells were washed with 1X Perm/wash buffer and fixed with 2% ultra-pure formaldehyde (Life Technologies). Analysis was performed in triplicate and analyzed using ImageStreamX IDEAS 6.2 software (Millipore) after 100,000 cells were collected.