Epithelial Cell Invasion Assay for Candida albicans

C. albicans invasion of epithelial cells was determined as described previously43 (link). Briefly, TR146 epithelial cells were grown to confluence on glass coverslips for 48 h and then infected with C. albicans yeast cells (1×10⁵), for 3 h in a humidified incubator (37 C/5% CO₂). Following washing (3x PBS), the cells were fixed with 4% paraformaldehyde. All surface adherent fungal cells were stained for 1 h with a rabbit anti-Candida antibody and subsequently with a goat anti-rabbit-Alexa Fluor 488 antibody. After rinsing with PBS, epithelial cells were permeabilized (0.1% Triton X-100 in PBS for 15 min) and fungal cells (invading and non-invading) were stained with Calcofluor White. Following rinsing with water, coverslips were visualized using fluorescence microscopy. The percentage of invading C. albicans cells was determined by dividing the number of (partially) internalized cells by the total number of adherent cells. At least 100 fungal cells were counted on each coverslip.