Isolation and Assay of Innate Immune Cells

For the preparation of exudate neutrophils and peritoneal macrophages (Mphs), C57BL/6 mice were ip-injected with 0,5 ml 10% proteose peptone (Sigma, St. Louis, MO, USA)/PBS. After 4 h (for neutrophils) or 3 days (for peritoneal macrophages), the peritoneum was flushed with 7 ml PBS containing 50 U/ml heparin to collect cells. For isolation of resting Mphs, peritoneum of untreated mice was flushed. For isolation of bone marrow resident neutrophils, bone marrow was collected from mice. After lysis of red blood cells, samples were separated on a discontinuous Percoll gradient: from bottom to top 78%, 69%, and 52% Percoll (GE Healthcare). Gradient was centrifuged at 1500×g and 4°C for 30 min. Cells accumulating in the interphase between the 78% and 69% Percoll layer were collected as neutrophils. All different types of immune cells were plated in RPMI supplemented with 10% heat-inactivated FCS and used for co-culture with fungi.
For the in vitro Ca killing assay, innate immune cells were plated in replicates at a density of 1×10⁵ cells/well of 96-well plates. Cells were incubated with Ca at indicated MOIs and for indicated time. After incubation, mammalian cells were lysed by addition of Triton X 100 to a final concentration of 1%. After lysis, wells were extensively scrapped, 2× washed with PBS and surviving Ca was determined by plating serial dilutions of the collected media and washes in duplicates on YPD plates containing ampicillin (Sigma). The percentage of killing was calculated according to the following formulas (df = dilution factor):