Live-cell Imaging of C. albicans-Induced Cell Death

Live-cell imaging of the cell death dynamics using PI staining was performed as described previously (78 (link)). In brief, a confluent monolayer of HEK293A cells in a 24-well plate was washed once with culture medium and subsequently infected with 1 × 10⁵C. albicans yeast cells in culture medium or culture medium containing human albumin (Albuman; Sanquin Plasma Products B.V.) concentrations between 10 mg/ml and 0.1 mg/ml. All media contained 4 μg/ml propidium iodide (Sigma-Aldrich). Cells were imaged in a Zeiss Celldiscoverer 7 for 18 h at 37°C and 5% CO₂. Microscopy pictures were taken every 20 min at 10 × magnification from four independent positions per well in the bright field, as well as fluorescence with excitation at 353 nm, and emission was recorded at a wavelength of 465 nm.
Microscopy pictures from the red fluorescence channel were analyzed using Fiji (79 (link)). Using the threshold function, images were converted to binary images, and the number of PI-positive nuclei was enumerated for each image using macro batch analysis and the Particle Analyzer tool. Based on the obtained counts of PI-positive kidney cells, the percentage of dead kidney cells was calculated in relation to the maximal number of dead cells.