Cell wall staining and flow cytometry.

For chitin and exposed chitin detection, cell wall staining with wheat germ agglutinin (WGA) and calcofluor white (CFW) was assessed as previously described (32). Briefly, overnight YPD cultures were diluted 1:10 in CO₂-independent liquid medium and incubated (∼18 h) at 37°C with 150-rpm shaking. Cells were stained with 100 μg/ml of fluorescein isothiocyanate (FITC)-conjugated WGA and 25 μg/ml CFW and incubated in the dark for 35 min and 10 min, respectively. Quantitative analysis using ImageJ software was performed as previously described (32, 41).

For flow cytometry analysis, cells were incubated similarly as described above and fixed with 3.7% formaldehyde for 5 min at room temperature. Cells were then slowly pelleted and washed twice with phosphate-buffered saline (PBS). Cells were stained with 100 μg/ml FITC-conjugated wheat germ agglutinin (WGA; Molecular Probes). Cells stained with WGA were incubated in the dark at room temperature for 35 min. Cells were then slowly pelleted and washed twice with PBS. Cells from each strain were stained and resuspended in PBS at a concentration of 10⁷ cells/ml. Cells at 10⁶/ml were submitted to the Duke Cancer Institute Flow Cytometry Shared Resource for analysis using a BD FACSCanto II flow cytometer. Data were analyzed by FlowJo v10.6.1 software (FlowJo, LLC). Unstained cells were used as negative controls, and positive events were gated in the forward scatter/side scatterplots and represented as histograms. Geometric means were calculated based on the mean fluorescence intensity (x axis of histogram) of all cells quantified for each strain (y axis of histogram).