Polystyrene 96 well plates

Anti-ovalbumin antibodies were evaluated by ELISA, as previously described (Thommen Maciel Sartor, Moleta Colodel & Albuquerque, 2011 ). Briefly, polystyrene 96 well-plates (Falcon) were coated overnight at 4 °C with 0.5 mg of OVA per well in carbonate-bicarbonate buffer pH 9.6, after that washed with PBS 0.05%-Tween-20, and then blocked with 1%-BSA/PBS for 1 h at room temperature. Mouse antiserum from day 45 was added by triplicate (serial dilutions from 1:100) and incubated overnight at 4 °C. Plates were washed and incubated for 1 h at 37 °C with goat anti-mouse IgG-HRP (Catalog No. sc-2005; Santa Cruz Biotechnology Inc., Dallas, TX, USA). After that wells were washed, and tetramethylbenzidine (TMB) was added. The reaction was stopped with H₂SO₄. Plate was read at 450 nm in a plate reader (Synergy HT; Biotec, Emmerich, Germany), values indicating optical density (DO). The antibodies isotypes (IgG1, IgG2a, IgG2b, IgG3, IgA and IgM) were determinate by commercial kit Ig Isotyping Mouse Uncoated ELISA (Catalog No. 88-50630; Thermo Fisher Scientific Inc., Vienna, Austria).

The minimal inhibitory concentration (MIC) was determined using a microbroth dilution method with an initial inoculum of early exponential bacteria. All test dilutions were made in TSB, to obtain a similar GAE content between test extracts (Table 1). Specifically, cells were grown to the early exponential phase of growth in TSB and cells (20 µL, approximately 2 × 10⁴ CFU per well) were incubated with increasing dilutions of test solutions in a final volume of 200 µL per well (Polystyrene 96-well plates (Falcon, Corning, NY, USA)) for about 18 h at 37 °C in air. The cell density was determined using a microtiter plate reader (BioTek, Winooski, VT, USA) at an optical density of 600 nm or 630 nm. The cell number was determined by plating samples on Tryptic Soy Agar (TSA) plates, incubating overnight at 37 °C, and colony-forming units (CFU) counted the next day. The MIC was taken as the lowest drug concentration resulting in observable colonies. The minimal bactericidal concentration (MBC) was taken as the lowest drug concentration that resulted in no observable colonies. All experiments were performed in triplicates. The optical density (OD) of test solutions in TSB (no cells) were determined and used as background values. The positive controls included growing cells in TBS alone or TSB with relevant solvents.