Opsys mr

BCAM and BHAM assays were performed as described previously [26 (link)]. In these assays, A. fumigatus grows out into biofilms covering the agar surface. Briefly, RPMI agar containing 2.5x10⁴ to 10⁵A. fumigatus conidia/ml agar (as specified for different experiments in the Results section) was distributed into sterile flat-bottom 96 well cell culture plates (COSTAR, Corning, NY) at 100 μl/well. Upon agar solidification, wells were either incubated at 37°C for 24 hours before loading (= BHAM assays), or immediately loaded with 100 μl of test substances (= BCAM assays). Control wells on each test plate contained 100 μl of RPMI 1640 medium, allowing the conversion of test results to % of the RPMI control (= 100%). Loaded plates were incubated at 37°C for 24 hours. Fungal metabolism was determined by XTT metabolic assay at 490 nm [40 (link),43 (link)]. Menadione (vitamin K3) was used as an ingredient in the XTT metabolic assay, boosting the reduction of tetrazolium salts to formazans. XTT assays were evaluated using a plate reader (Opsys MR, DYNEX Technologies, Chantilly, VA). Although XTT is a measure of metabolic activity of cells, previous studies of A. fumigatus have indicated that XTT results are linear with mass, and equated XTT result with dry weight [44 (link)–46 (link)].

β-Hexosaminidase release was measured as described previously (29 (link)). Mouse platelets were stimulated with increasing concentrations of thrombin at 37 °C for 10 min under non-stirring conditions and the supernatant added to 20 μl of 0.1 m 4-nitrophenyl n-acetyl-β-d-glucosaminide (Sigma) substrate in citrate-phosphate buffer (0.2 m Na₂HPO₄, 0.1 m citric acid, pH 4.2) in a 96-well plate. Total controls for β-hexosaminidase activity were obtained by repeated snap-freeze/thaw of the same volume of non-stimulated platelets. The reaction was quenched with NaOH after a 1-h incubation at 37 °C and absorbance was read at 405 nm (Opsys MR, Dynex Technologies, Worthing, UK). Secreted β-hexosaminidase was expressed as percentage of TOTAL.

Streptavidin plates (Thermo Fisher Scientific) were coated overnight at 4°C with 1 µg/well biotinylated RG1-peptide (JPT Peptide Technologies, Berlin, Germany) in coating buffer (0.1 M Tris/HCl pH7.4 + 0.15 M NaCl + 0.1% Tween-20). On the next day, plates were excessively washed with coating buffer and blocked with 1% milk/PBS for one hour. After washing, plates were incubated with four-fold serial serum dilutions (ranging from 1:100 to 1:102,400) for 1 h, washed again and incubated with an HRP-conjugated goat-anti mouse or anti-rabbit antibody (Bio-Rad) for 1 h. Plates were developed using ABTS (Roche) substrate and OD₄₀₅ was determined (Opsys MR, Dynex Technologies). The cut-off of 0.177 was calculated by measuring sera of virus-free animals and adding three standard deviations to the mean (0.131 + 0.046).

For probing protein–protein interactions, rDSP protein at the concentration of 300 μg/ml was dialyzed against 0.1 M NaHCO₃ and then reacted with 100 μg/ml Sulfo-NHS (N-hydroxysuccinimido)-LC (long-chain)-biotin (Pierce, Rockford, IL, USA) for 20 min at RT, followed by 2 h at 4 °C. Free biotin was removed by dialysis against 50 mM Tris-HCl and 150 mM NaCl, pH 7.4. To characterize the relative rDSP interaction with MMP9, substrate binding assay was performed as described earlier40 (link). Briefly, 96-microwell plates were coated with 1 μg/well of mut-rMMP9 or bovine serum albumin (BSA) as control overnight at 4 °C and non-specific binding were blocked with 1% BSA (Sigma-Aldrich, MO, USA) for 1 h at RT. After thorough rinses with PBS, the biotinylated rDSP ranged from 0–18 fold molar excesses in PBS with 1% BSA was added into the plates and incubated for 30 min. Bound rDSP was reacted with AP-conjugated streptavidin diluted 1:10,000 in PBS for different time points using 1 mg/ml PNPP (ρ-nitrophenyl phosphate disodium) as substrate (Pierce, Rockford, IL, USA) and quantified at 405 nm (Opsys MR, Dynex, Chantilly, VA, USA). BSA was used as control group and the binding of rDSP to mut-rMMP9 was expressed as relative binding activity compared to the control group. The experiments were performed three times in triplicate.

Cell viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) using 1× 10⁴ cells seeded in a 96-well microplate. The next day, the cells were exposed to curcumin (5 µM), andrographolide (14 µM), and their combination for 24 h. After the treatment, 20 µL of MTT (5mg/mL PBS) was added to each well and incubated at 37 °C for 4 h. After incubation, the media were removed and 200 µL of DMSO was added to dissolve formazan crystals by constant shaking for 15–20 min. The purple formazan was quantitated by measuring the absorbance at 540 nm with a reference wavelength of 630 nm by a microplate reader (OPSYS MR, Dynex Technologies, Chantilly, VA, USA). Cell viability was measured as the percentage of the optical density (OD) values of the treated cells compared with the untreated cells as the control.

The cytotoxic effects of analysed compounds were assessed by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT was purchased from Millipore Sigma (Milan, Italy). For this assay, 8 × 10³ cells/well were plated in a 96-well plate and incubated with compounds for periods reported in the Results section. Then, 10 μL of MTT solution (5 mg/mL in PBS) of added to the cell medium, and the incubation was protracted for 2 h at 37 °C in the dark. MTT is metabolised to formazan salt in viable cells [55 (link)]. Afterwards, the media were taken out, and cells were lysed in lysis buffer (20% SDS and 10% dimethylformamide). The cell viability was determined by analysing the absorbance of the formazan read at 490 nm with 630 nm as a reference wavelength using an automatic ELISA plate reader (OPSYS MR, Dynex Technologies, Chantilly, VA, USA). IC₅₀ values were determined by Graphpad Prism 7.0 software (San Diego, CA, USA).