2.5 Cytotoxicity analysis
The Vero cell lineage, established from the kidney cells of the African green monkey (Cercopithecus aethiops ) (CCIAL 057, Adolfo Lutz Institute, São Paulo), was used for the in vitro experiments. The Vero cells were cultured in Ham F-10 medium (Sigma-Aldrich), with fetal bovine serum (10%; Sigma-Aldrich) and 100 μg/mL penicillin/streptomycin, and the culture was maintained at 37 °C with 5% CO2. Assays were performed in triplicate, according to ISO10993-5 (2009).
For this assay, the antibodies were diluted in Ham F-10 medium (Sigma-Aldrich) with concentrations ranging from 2 mg/mL to 0.0625 mg/mL.
For the MTT assay, the Vero cells were seeded in 96-well plates (Corning) at a density of 1.5 x 106 cells/well. After 24 hours, the culture medium was removed and substituted with 100 µL of the diluted antibody solution. The plate was incubated for 24 hours, and then, the cell morphology was analyzed. The analysis was performed with phase contrast in an inverted microscope (Axiovert A1 Axioscope, Zeiss).
After the morphologic analysis, 100 µl of 10% MTT solution (5 mg/ml, Sigma-Aldrich) in phosphate-buffered saline was added to each well, and the cells were incubated for 2 hours at 37 ºC. After removing the MTT solution, 50 μL of dimethyl sulfoxide (Synth) was added to each well for 10 minutes, and the absorbance was measured with an automated spectrophotometric microtiter plate reader (SpectraMax M5) using a 570 nm filter.