2.4.3. Cell Viability by MTT

The MTT assay was performed using cell cultures at T₀, T₂₄, T₄₈, T₇₂, T₉₆, and T₁₄₄ and according to the methodology described by Mosmann and modified by Carvalho et al. [38,39]. Briefly, the MTT solution (at a final concentration of 400 µg mL⁻¹ per well) was added to 96-well plates prepared previously with 104 cells/well followed by incubation at 16 or 18 °C (depending on fish species) for 4 h. Following the incubation period, the medium with the MTT was removed and the formazan crystals dissolved using a 150 μL/well 4:1 mixture of dimethyl sulfoxide (DMSO)/glycine buffer (50 mM glycine, 50 mM sodium chloride/NaOH, pH 10.5) by shaking for 20 min at 200 rpm. The absorbance was measured at 550 nm on a microplate reader. Cell viability was expressed as the percentage of T₀ cells.