Multiskan plate reader

For the monitoring of the antibiofilm properties of ResNPs, SeNPs, and composite, the preparation of bacteria was conducted as in minimal inhibitory concentration assays, but 10× more bacteria were seeded to every well containing various concentrations of samples. Glucose was added up to 2% to promote biofilm formation. Bacteria were then incubated overnight, followed by the removal of the medium and washed 3 times with 1xPBS to remove all non-adherent bacteria. The researchers added 0.1% crystal violet solution to the wells and incubated for 20 min. Then, the plates were rinsed with fresh water to remove excess stains and thoroughly dried. The stain left bonded to biofilms was dissolved by using absolute ethanol, and absorbance was recorded at 570 nm using a Multiskan plate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Adhesion to abiotic surface (polystyrene) was analyzed using 96-well plates as described previously [26 (link)]. Overnight cultures of bacteria grown in LB broth (10 μl) were added to 1 ml of LB. This volume was distributed in quintuples (200 μl per well) into a 96-well plate and incubated at room temperature for 24 h. Unbound bacteria were removed by washing the wells three times with PBS, and bound bacteria were stained with 1% violet crystal (CV) for 20 min. Wells were thoroughly rinsed thrice with PBS, and the dye in the adhered bacteria was solubilized with 100 μl of ethanol 70%. Finally, the amount of extracted violet crystal was determined using an enzyme-linked immunosorbent assay (ELISA) and measuring the OD₆₀₀ in a multiskan plate reader (Thermo Scientific).

MRC-5 pd19 cells were seeded onto 96-well flat-bottomed plates at a concentration of 10,000 cells/well. TiO₂, TiO₂-GA or GA was suspended/dissolved in the completed culture medium at the final concentrations of 0.01%, 0.10% and 1.00% at the final volumes of 200 µL per well. After 24 h, each of the reagents was added to the cells. Control cells were not treated with synthesized reagents; they were incubated with completed cell culture medium. The incubation of cells with TiO₂, TiO₂-GA or GA lasted 24 or 48 h. Then, the medium was discarded, and the new medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Affymetrix, Cleveland, OH, USA) at a final concentration of 0.5 mg/mL was added to the cell culture. Cells were incubated for 2.5 h in cell culture conditions. Next, the medium was removed, and 100 µL of DMSO (Thermo Scientific, Waltham, MA, USA) was added to dissolve the formed formazan crystals. The absorbance was read with a Multiskan plate reader at 570 nm, background 655 nm (Thermo Scientific, Waltham, MA, USA). The experiment was performed in triplicate.

HK activity was measured using a hexokinase assay kit (ScienCell Research Laboratories, Carlsbad, CA, USA) according to the assay procedures recommended by the manufacturer. Purified hexokinase was pre-incubated with various concentrations of AF for 10 min, and then mixed with the assay substrates to start the reaction. The changing NADPH absorbance was monitored in dark for 60 min, using a Multiskan plate reader (Thermo Scientific). For analysis of HK activity in cell lysates, A549 cells were treated with various concentrations of AF for 6 h, and protein extracts were prepared and immediately used for assay of HK activity using the substrate mixtures provided in the assay kit.

Cytotoxicity was measured using MTS assay. Cells were seeded in a 96-well plate (2000 cells per well) and incubated with AF and ADM for 72 h. Overall, 20 µL MTS solution was then added to each well and incubated for another 4 h at 37 °C. The optical density (OD) at 490 nm was determined using a Multiskan plate reader (Thermo Scientific, Helsinki, USA). Cell survival rate was calculated as the proportion of survival cells after a drug treatment relative to the untreated control cells.