Synergy spectrophotometer

The total phenolic content (TPC) was measured according to the Folin-Ciocalteau procedure [62 (link)]. Samples (0.25 and 0.125 mg) were mixed with Na₂CO₃ (2.25 mL; 7.5% w/v) and Folin-Ciocalteu reagent (0.25 mL) and allowed to stand for 3 h at room temperature. The absorbance was read at 765 nm using a Synergy spectrophotometer (Biotek, Winooski, VT, USA). Data were expressed as milligrams of gallic acid equivalents (GAEs) per g of extract. To this purpose, a gallic acid calibration curve (y = 0.0247x − 0.0063; R² = 0.9998) was built up in the range 0.78–25 µg/mL (final concentration levels).

An enzyme-linked immunosorbent assay was used to measure concentration of soluble intercellular adhesion molecule-1 (sICAM-1) (R&D Systems, cat. no DY720-05) and von-Willebrand factor (vWF) (R&D Systems, cat. no DY2764-05) from perfusate samples. Standards for each plate were used to generate a curve. Perfusates were diluted to 1:1000 for sICAM-1 and used neat for vWF. Standards and perfusate samples were applied in technical duplicate. Optical density of plates was assessed through the Synergy spectrophotometer and Gen5 plate reading software (BioTek) at 450 nm. Final concentration values were adjusted to TLC.

The total phenol content (TPC) was determined according to the Folin-Ciocalteau procedure [29 (link)]. Samples (0.25 mg and 0.125 mg) were mixed with 2.25 mL of Na₂CO₃ (2.25 mL; 7.5% w/v) and 0.25 mL of Folin-Ciocalteu reagent. The tubes were mixed and allowed to stand for 3 h at room temperature (T = 25 °C), in the dark. The absorbance was read at 765 nm using a Synergy spectrophotometer (Biotek, Winooski, VT, USA). Data were expressed as milligrams of gallic acid equivalents (GAEs) per g of extract. To this purpose, a gallic acid calibration curve ( $y=0.0247x-0.0063$ ; R² = 0.9998) was built up in the range 0.78–25 µg/mL (final concentration levels).

The total condensed tannin (TCT) content was determined according to Butanol-HCl method [32 (link)], with some modifications. The samples (0.1 mL) were dissolved in 3 mL of the Butanol-HCl reagent (95:5, v:v; HCl concentrated 37%) and 0.1 mL of the ferric reagent (2% w/v in 2 N HCl) was added. The tubes were mixed and put in a heating block adjusted at 97 °C for 60 min. The absorbance was read at 550 nm against the blank (water) using a Synergy spectrophotometer (Biotek, Winooski, VT, USA). Data were expressed as milligrams of cyanin equivalents (CYEs) per g of extract. To this purpose, a cyanin calibration curve ( $y=0.0339x-0.1202$ ; R² = 0.9946) was built up in the range 1.95–62.5 µg/mL (final concentration levels).

Total phenol content (TPC) was determined according to the Folin–Ciocalteau method, as well as the surface phenol content. Briefly, for TPC, 2.5 and 5.0 mg of FE-MD were mixed with 2.25 mL of sodium carbonate (7.5% w/v) and 0.25 mL of Folin–Ciocalteau reagent. The mixtures were shaken for 3 h in the dark at room temperature. The absorbance was read at 765 nm using a Synergy spectrophotometer (Biotek, Winooski, VT, USA). Three replicates of three aliquots (n = 3) of each sample (in total, 3 × 3 measurements) were analyzed. A calibration curve of gallic acid was built up (0.78–50 µg/mL) and data were expressed as mg of gallic acid equivalents (GAEs) per g of sample (mean ± SD). For surface phenol content, FE-MD powder was mixed with ethyl acetate (1.0 mL), vortexed for 60 s, and centrifuged at 3000× g for 10 min. The surface phenol content was measured following the same Folin–Ciocalteau method. The embedded phenol content was calculated by subtracting the surface phenol content from the TPC value [48 (link)].

Candida species were grown as described above, washed, and diluted to 10⁵ cells/mL in yeast nitrogen base (YNB) medium containing 0.5% oyster glycogen (Sigma-Aldrich, G8751), bovine liver glycogen (Sigma-Aldrich, G0885), or glucose. 200 μL were transferred to the wells of a microtiter plate. OD₆₀₀ readings were captured at 60 min intervals, using a BioTek Synergy spectrophotometer with an incubation temperature of 30°C and orbital shaking at 200 rpm. The experiments were repeated in technical quadruplicate and biological triplicate. The data are expressed as the mean ± standard error of the mean (SEM). For the stress tolerance and phenotypic assays, C. albicans overnight cultures were washed twice with PBS and adjusted to 2 × 10⁷ cells/mL. Serial dilutions of cells were spotted (5 μL) onto YP agar plates containing dextrose (or other carbon sources, as indicated) under the following conditions: temperature (30°C, 37°C, or 42°C), pH (3.5 and 8.5), peroxide stress (5 mM or 25 mM H₂O₂), osmotic stress (1 M sorbitol), ionic stress (100 or 500 mM CaCl₂, 1.5 M NaCl), metal ion stress (10 mM MnCl₂), cell wall stress (25 μg/mL Congo red, 0.05% sodium dodecyl sulfate), or alternative carbon sources (3% glycerol or 3% ethanol). Unless indicated, the plates were incubated at 30°C for 48 h, prior to the acquisition of images using a Gel Doc XR Imaging System (Bio-Rad).