Multiscan ex elisa plate reader

The biofilm-forming ability of the S. aureus strains was studied in 96-well microtiter plates using TSB broth in the presence of compounds. The biofilm-forming ability of the E. coli strains was studied in 96-well microtiter plates using LB broth in the presence of the compounds. The overnight cultures of the bacteria were diluted to an OD of 0.1 at 600 nm and then added to each well, with the exception of the medium control wells, and compounds were added at an MIC/2 concentration (where the MIC was >100 µM, the tested concentration was 50 µM or 100 µM). The final volume was 200 μL in each well. The positive control was CCCP at 50 µM. Plates were incubated for 48 h at 30 °C with gentle shaking at 100 rpm. After the incubation period, the medium was removed and the plate was washed with water to remove unattached cells. A total of 200 μL CV (0.1% [v/v]) was added to each wells and incubated for 15 min at room temperature. CV was discarded from the wells and the plate was washed with water again. Then, 200 μL of 70% ethanol was added to each well and the biofilm formation was determined by measuring the OD at 600 nm using a Multiscan EX ELISA plate reader (Thermo Labsystems, Cheshire, WA, USA). The antibiofilm effect of the compounds was expressed as the percentage (%) decrease in biofilm formation.

The ability of selected compounds to decrease the formation of biofilm was also studied in two strains of S. aureus: S. aureus ATCC 25923 and S. aureus 272123. Biofilm formation was detected using crystal violet (CV), a dye, at 0.1% (v/v). The initial inoculum was incubated overnight in TSB, and diluted to an OD₆₀₀ of 0.1. Then, the bacterial suspensions were transferred to 96-well microtiter plates, and the compounds were added at a subinhibitory concentration (half the MIC or 100 µM if the compound did not present an observable MIC for these strains in the previous assay) to a final volume of 200 µL. The EPI reserpine served as the positive control.
The plates were incubated for 48 h at 30 °C with stirring at 100 rpm. When this incubation period was over, the incubation medium was disposed of, and the unattached cells were removed by rinsing the plate with tap water. Then, a volume of 200 µL of a 0.1% (v/v) CV solution was added, followed by a 15 min incubation at room temperature, after which the CV solution was discarded, and the plates rinsed tap water again. Finally, 200 µL of ethanol (70%) were added to the wells.
The OD₆₀₀ was measured using a Multiscan EX ELISA plate reader (Thermo Labsystems, Cheshire, WA, USA), and the effect of the compounds in the formation of biofilm was expressed in percentage (%) of decrease in biofilm formation.

Ninety-six wells Maxisorp microtiter plates (NUNC) were coated with 100 μL/well containing 30 μg of serum or thymic protein extracts of wild type or Tat-Tg mice, and incubated over-night at 4°C. The following day, the plate was washed and blocked in 200 μL of blocking buffer (PBS 1 × , 5% non-fat dry milk, 0.01% tween 20) and next incubated in the same buffer in presence or absence of anti-Tat antibody (HIV-1 Tat mAb 5A5.3) (1 μg/mL) for 2 h at 37 °C. Next, each well was washed six times with PBS 1 × containing 0.05% tween 20, and then incubated with a 1:5000 dilution of Alkalyne Phosphatase conjugated goat anti-mouse IgG for 1 h at 37 °C. After extensive washing, samples were incubated with PNPP (Para-Nitro-Phenyl-Phosphate) platelets substrate, diluted in Diethanolamine substrate solution pH 9.6, for 10 min at room temperature. Optical density (OD) was measured twice at 420 nm on a MULTISCAN EX ELISA Plate reader (Thermo Labsystems, Franklin, MA).