Costar 3603

A CytoTox 96^® Non-Radioactive Cytotoxicity Assay (Promega G1781) was performed according to the manufacturer’s instructions. Briefly, Substrate Mix was dissolved in 12 mL Assay Buffer. A total of 30 μL of cell supernatant was transferred to a 96-well assay plate (Corning Costar 3603) and combined with 30 μL of substrate solution. The plate was incubated at RT for 30 min in the dark. After that time, 30 μL of Stop Solution was added to each well. The absorbance was measured at 490 nm using a BMG LABTECH CLARIOstar plate reader. Samples were assayed in duplicate.

C. albicans isolates with LBF (n = 3), HBF (n = 3), CA14, and chitinase mutants Δcht2, Δcht3 and Δcht2/Δcht3 biofilms were grown in RPMI-1640 for 4 and 24 h at 37°C. The quantity of eDNA release was measured using a microplate fluorescence assay (MFA) using a DNA binding dye (SYBR® Green I), as previously described [14 (link)]. Briefly, SYBR® Green I (Invitrogen) was added to biofilm supernatants in a black well microtitre plate (Costar3603; Corning) at a ratio of 1:4. Binding of this dye produces fluorescence directly proportional to DNA concentration. The levels of eDNA were quantified using a fluorescence plate reader (Fluostar Optima; BMG Labtech) at 485 and 518 nm, respectively. The concentration of eDNA in the sample was quantified using the DNA standard curve as previously described [29 (link)]. In addition, optical density of the culture was measured at 530 nm simultaneously for normalising the relative fluorescence units (RFU) data. Each isolate was tested in duplicate, on three separate occasions.

The pSB1C3 plasmids harboring the psicose biosensors were introduced into E. coli DH5α. Transformed cells were grown overnight at 37°C in LB medium containing 35 µg/ml chloramphenicol. The suspension was diluted by 100 in the same medium and incubated at 37°C and 200 rpm for 1 h. Afterwards, a 96 well plate (COSTAR^® 3603, Corning Inc.) was prepared and each well was filled with 120 µl of cell suspension and 30 µl of a solution containing Psicose and IPTG. Different concentrations of Psicose (0, 0.1 µM, 1 µM, 10 µM, 100 µM, 1 mM, 10 mM, 100 mM, 200 mM and 300 mM) and IPTG (0, 1, 10, 100 and 1000 µM) were tested. The plate was incubated at 37°C at 200 rpm, fluorescence and OD_600nm were measured every 7 min during 150 cycles. Fluorescence of mCherry was measured using CLARIOstar^® plate reader (BMG Labtech) at 587/610 nm, the mCherry wavelengths of fluorescence excitation and emission (36 (link)). Fluorescence of mEmerald was measured using Synergy™ HTX plate reader (BioTek^® Instruments, Inc.) at 485/528 nm, the mEmerald wavelengths of fluorescence excitation and emission (37 (link)). The experiments were performed in triplicate and the fluorescence values (background subtracted) normalized by cell density (OD_600nm).