Journal: Journal of Fungi

Article Title: Calcineurin Inhibitors Synergize with Manogepix to Kill Diverse Human Fungal Pathogens

doi: 10.3390/jof8101102

Figure Lengend Snippet: MGX treatment increases chitin levels and exposes (1→3)-β-D-glucan at the fungal cell surface. ( A ) C. albicans was grown in YPD supplemented with MGX for 24 h at 30 °C then growth was measured by optical density. Left: Mean relative growth for duplicate wells from a representative experiment. Cells were formaldehyde fixed, then stained with CFW or labelled with a mouse monoclonal antibody to (1→3)-β-D-glucan and goat anti-mouse secondary antibody conjugated to AlexaFluor488 and examined by flow cytometry. Right: Median fluorescence intensity for CFW and AlexaFluor488 labelled cells normalized to untreated controls (>20,000 events/sample). Fluorescence intensity for CFW staining saturated the flow cytometer at [MGX] > 31.25 ng/mL. ( B ) Representative fluorescence micrographs of CFW-stained C. albicans (blue) prepared as in ( A ). Scale bar, 20 µm. ( C ) Representative (1→3)-β-D-glucan immunofluorescence (green) micrographs of C. albicans prepared as in A. Scale bar, 20 µm.

Article Snippet: Cells were immunostained by addition of 100 μL anti (1→3)-β-D-glucan monoclonal antibody (Biosupplies Australia) diluted at 1:500 in PBS-BSA containing and then incubated at RT for 1 h. Cells were washed 3× with 100 μL PBS supplemented with 0.05% ( v/v ) Tween 20 (PBS-T).

Techniques: Staining, Flow Cytometry, Fluorescence, Immunofluorescence