Spectramax id5 reader

The binding affinity between filipin and C. albicans cells was measured as described previously (Ren et al. 2014 (link); Zhu et al. 2021 (link)). C. albicans were cultured in RPMI 1640 medium at a final concentration of 5 × 10⁴ CFU/ml and treated by sIgA (0, 50, and 100 µg/ml) for 2 and 4 h. C. albicans cells were collected and rinsed by PBS and then treated with 20 µg/ml filipin at 37 °C, 200 rpm for 30 min. The cultures were then collected and washed by PBS. SpectraMax iD5 reader (Molecular Devices, LLC., San Jose, America) with excitation at 340 nm and emission at 480 nm was employed to measure the fluorescence.

Inhibitors were dissolved in dimethylsulfoxide (DMSO) at 10 mM and dispensed to final concentrations in 384-well plates (Corning) using Echo 550 Liquid handler (Labcyte) or D300e digital dispenser (Tecan). Cells were seeded at 1,000 (SW620) and 500 (HCT116) cells per well in 50 µl of complete medium. Plates were incubated in a humidified chamber for 96 h at 37 °C and 5% CO₂. Cell viability was determined by adding 10 µg ml⁻¹ resazurin (Sigma Aldrich) and measuring conversion to resorufin after 5 h. Fluorescence at 595 nm was measured with Hidex Sense reader and Hidex Sense Software v.1.0 (Hidex Oy) or SpectraMax iD5 reader (Molecular Devices). Half-maximal effective concentration (EC₅₀) was calculated with logistic nonlinear regression in Prism v.9.3.1 (GraphPad).