Evaluation of Cell Wall Damage by 2-Chalcone

Confocal microscopy was used to evaluate the damage to the cell wall caused by 2-chalcone, using fluorochrome Calcofluor White (Thermo Fisher Scientific) and T. rubrum ATCC 28189. Fungal suspensions were prepared at a concentration of 1 × 10⁶ cells/mL and added to 24-well plates containing sterile coverslips, along with sub-inhibitory doses of 2-chalcone (7.8 mg/L). After the incubation period (35°C, for 96 hours), the supernatant was removed and the coverslips were washed with PBS. The coverslips were covered with Calcofluor White solution (100 mg/L), and the plates were incubated again at 37°C, for 45 minutes, protected from light. Then, the coverslips were washed with PBS, removed from the wells, and mounted on 4 μL of Fluoromount-G (Sigma-Aldrich), which was previously deposited on microscopic slides. The slides were then observed using a confocal microscope (Carl Zeiss LSM 800 with Airyscan) with an image capture and processing program (Software ZEN BLUE 2.3 System) at the Faculty of Dentistry, UNESP-Araraquara, Brazil (Curcio et al., 2017 (link); Oliveira et al., 2020 (link)).