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.