Cellstar tissue culture plate

In a 12-well plate (Cellstar^® tissue culture plate, Greiner Bio-One, Cassina de’ Pecchi, Italy), 4 × 10⁵ cells/well were seeded and left to incubate for 24 h. The medium was aspirated and the antioxidant samples were introduced, consisting of either 1 mL of α-tocopherol in a CSO sample [29 (link)] at the final α-tocopherol concentration of 10 µM or 1 mL of CS-CO 100 diluted to 1:20 (v/v) with the medium. Both the samples were left in contact with the cells for 3 h. A negative control consisting of untreated cells (100% damage) was also evaluated. The samples were removed and the damage was induced with 1 mM H₂O₂ for 1 h. Each well was washed with 1 mL of PBS and then 1 mL of an 18 mg/mL α-phenyl-N-tert-butyl-nitrone (PBN) spin trap solution was added and left for 30 min. The PBN solution was removed, a further washing of the wells was carried out, and the cells were subject to trypsinization to detach them from the bottom of the plate. After centrifugation, the supernatant was eliminated and for each analysis, the pellet obtained by joining two wells was used, taken up with 200 µL of Hank’s balanced salt solution (HBSS). The analysis was conducted with an EPR spectrometer (BRUKER EMX/12 with an ER4102ST cavity and a window for UV/Vis radiation, Bruker, Milan, Italy).

Previously described methods were used (50 (link), 51 (link)). Briefly, an overnight NTHi broth culture was diluted 1:200 in fresh broth and 200 μL was inoculated into the wells of a 96-well CELLSTAR tissue culture plate (Greiner Bio-One, Monroe, NC). The plates were incubated at 37°C on a nutator for 24 h. Before biofilm quantitation, growth was assessed by measuring the OD₄₉₀ in a Bio-Rad plate reader. To quantitate biofilm formation, 20 μL of Difco crystal violet (Becton, Dickinson and Co., Sparks, MD) was added to each well and incubated at room temperature for 15 min. Wells were washed vigorously with distilled water and the plate was air-dried. A 230-μL volume of 95% ethanol was added to each well and the OD₅₇₀ was measured. Each plate included four control wells which contained sterile broth instead of bacteria but were otherwise treated identically. The OD₅₇₀ was standardized against these wells. Strains were tested in quadruplicate wells. The mean and standard deviation were calculated from values for each strain. Comparison of biofilm formation between mutant and parent strains was performed using an unpaired t test.