Fc microplate photometer

The cell viability was determined using the MTT assay [71 (link)]. The cells were plated in 96-well plates (3–5 × 103/well) for 12 h for adhesion and exposed to 200 μM Carvacrol for 24 hours. Then, cells were incubated with 20 μL of 5 mg/mL MTT (Abcam, Cambridge, UK) for 2 h, and the resulting formazan crystals were dissolved in 200 μL DMSO. The A490 was measured using the Thermo Scientific™ Multiskan™ (Waltham, MA, USA) FC Microplate Photometer. All data were normalized to the vehicle control or the negative control (NC) group.

The cells were plated in 96-well plates (3–5 × 10³/well) for 12 h and were treated as described before [42 (link)]. Then, cells were incubated with 20 μL of 5 mg/mL MTT (Abcam, Cambridge, UK) for 2 h and the resulting formazan crystals were dissolved in dimethyl sulfoxide (200 μL). Absorbance was measured at 490 nm using the Thermo Scientific™ Multiskan™ (Waltham, MA, USA) FC Microplate Photometer. All the data were normalized with vehicle-treated control cells. The experiment was performed at least in triplicate and repeated three independent times.

The T. gondii-specific IgG, IgG1 and IgG2a antibody levels in the serum samples were measured using enzyme-linked immunosorbent assay (ELISA) as previously described (30 (link)). Briefly, 50 μg of the recombinant proteins, i.e., HBc_Δ, H82, HBc_ΔH82, H301, HBc_ΔH301, R82, HBc_ΔR82, R301, and HBc_ΔR301, or 50 μl PBS (blank control) was adsorbed overnight onto 96-well plates (Corning Incorporated, Corning, NY) at 4°C in 50 mM carbonate buffer (pH 9.6). After blocking with 1% low fat milk in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST) for 1 h at 37°C, the mouse serum was diluted in PBS (1:25) and incubated at 37°C for 1 h. After washing three times with PBST, the anti-mouse-IgG, IgG1 or IgG2a HRP-conjugated antibodies (Sigma-Aldrich) were added. Peroxidase activity was detected using 10 mg/ml 3,3′,5,5′-tetramethylbenzidine (TMB; Sigma-Aldrich) and stopped by adding 50 μl 2M H₂SO₄. The results were recorded as the absorbance at 450 nm and detected with a Thermo Scientific Multiskan (Thermo, Waltham, MA) FC Microplate Photometer. All assays were performed in triplicate.