24 well microplate

HEK 293 cells (human embryonic kidney, ATCC no. CRL-1573) were cultured with high glucose in Dulbecco’s Modified Eagle Medium (DMEM) (EuroClone, Paington, UK) supplemented with a 15% fetal bovine serum (EuroClone, Paington, UK), 4 mM L-Glutamine (Sigma-Aldrich, St. Louis, MO, USA), 1% of the penicillin-streptomycin solution (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in a 5% CO₂ atmosphere.
For the propionate treatment, 0.5 × 10³ cells/mm² were seeded into the wells of a 24-well microplate (Costar, Corning Inc., Corning, NY, USA). After 24, 48, and 72 h, the medium was replaced with a fresh medium containing 10, 25, 50 mM of sodium propionate (Sigma-Aldrich, St. Louis, MO, USA) and cells were analyzed in the time-course by MTT and Neutral Red assays (see below) after 24, 48, and 72 h. The values were normalized versus untreated cells at respective time points.

AgNPs₁₀/No PEG, AgNPs₁₀/HO–PEG_9k–OH, AgNPs₁₀/MeO–PEG_9k–OMe, AgNPs₁₀/HS–PEG_9k–OMe, and AgNPs₁₀/c-PEG_9k were prepared in the same manner as the cell viability assay with the final concentrations of AgNPs (1.0 μg mL⁻¹) and of PEG (0.25 mg mL⁻¹) in DMEM (FBS (−)). HeLa cells were seeded at a concentration of 8 × 10⁴ per well in a 24 well microplate (Corning) and grown for 24 h. The cells were treated with 250 μL of AgNPs₁₀/No PEG, AgNPs₁₀/HO–PEG_9k–OH, AgNPs₁₀/MeO–PEG_9k–OMe, AgNPs₁₀/HS–PEG_9k–OMe, or AgNPs₁₀/c-PEG_9k followed by incubation for 2 h at 37 °C in 5% CO₂. After incubation, the cells were washed with D-PBS (−) and scratched using a pipette tip followed by washing with D-PBS (−). The medium was replaced with 100 μL of fresh DMEM (FBS (+)). The scratched regions were observed using a microscope (BZ-X800, Keyence). Subsequently, the cells were incubated for 22 h at 37 °C in 5% CO₂, and the scratched regions were measured again.

The minimum biofilm inhibitory concentration assay (MBIC assay) was performed by MTT staining assay. In this assay, bright yellow MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; Himedia] could be reduced by activated succinate dehydrogenase in viable cell mitochondria to blueviolet formazan, which was read at 550 nm after being dissolved in DMSO. The intensity of the color was correlated to the number of viable cells in remaining biofilm (Nair et al. 2016 (link)). In brief, an overnight culture of each strain was diluted in LB broth with 0.5% glucose to achieve ~ 5 × 10⁶ CFU/mL. Diluted cultures with various concentrations of BU were added (1 mL/well) into a 24-well microplate (Corning, USA), incubated at 37 °C for 24 h. After incubation, the wells were rinsed twice by 1 × PBS to remove planktonic and non-adhering cells. MTT staining was conducted as previously described (Nair et al. 2016 (link)). The experiments were performed in triplicates and repeated three times. The MBIC was defined as the minimum concentration of BU that prevents biofilm formation, indicated by no color development.

Microconidia were treated with AE‐mediated aPDT as described above. The pellets were centrifuged, washed with PBS and transferred into a 24‐well microplate (Corning, USA) containing round sterile coverslips. After incubation at 28°C for 2 h, the coverslips were carefully washed and immersed in 2.5% glutaraldehyde and kept at 4°C for 3 h. Following gentle wash with PBS, the microconidia were treated with 1% osmium tetroxide at 4°C for 3 h. Next, the samples on coverslips were dehydrated with 10–100% ethanol, freeze‐dried and sputter‐coated with gold. The prepared samples were finally viewed on a field emission scanning electron microscope (GeminiSEM 500; ZEISS, Thuringen, Germany). The microconidia without any treatment were used as control.

The inhibition of static biofilm formation was tested as previously described (Stepanovic et al., 2007 (link); Nair et al., 2016 (link); Yin et al., 2019 (link)). After overnight cultivation, 100 μl/well of the bacterial culture, diluted in LB broth with 0.5% glucose (about 5 × 10⁶ CFU/ml), was aliquoted into 96-well microplates (Corning, United States) with 1 μl of different concentrations (20 or 5 μg/μl) of each compound or DMSO and incubated at 37°C for 24 h. Using the same method, 1 ml/well of the diluted culture was added into 24-well microplates (Corning, United States) with 2 μl of compounds (100 or 25 μg/μl) or DMSO. After incubation, each well was rinsed with 1 × PBS to remove non-adherent cells and then dried at 37°C. CV staining was used to determine the remaining total biofilm biomass. Biofilms were stained with 0.5% CV for 10 min before washing. The washed biofilm mass was then dissolved in 30% acetic acid, and then the absorbance was measured at 550 nm.

Binding of fluorescent recombinant EGFP-LIC10778_Cards protein to cells was performed in VERO and pulmonary A549 cells. Confluent cultures were added to 24-well microplates (Corning®) at 2 × 10⁵ cells/well in complete DMEM (supplemented with antibiotics and 10% FBS) and incubated at 37°C, 5% CO₂. After 24 h of incubation, cells were washed once with PBS and incubated in serum-free DMEM for 5 h. Duplicate wells of adherent cells were incubated with 10–15 μg/well of recombinant EGFP-LIC10778_Cards samples or EGFP alone as control diluted in serum-free DMEM supplemented with 1% BSA for 2 h at 37°C under 5% CO₂. Unbound proteins were washed with PBS buffer containing calcium (132.5 mg/L) and magnesium (100 mg/L) and cells were fixed with 4% paraformaldehyde in PBS for 30 min at RT. After a washing step with the same PBS buffer, wells were further incubated with DAPI dye for nuclear staining. After final wash, cells were conserved in 90% glycerin solution for fluorescence evaluation, using EVOS M5 fluorescence microscope.