Spectrofluorometer

The measurement of MMP was as described elsewhere (Adeyemi et al. 2017 (link)). Briefly, growing HFF cells were treated with C. gigantea oil in the absence/presence of T. gondii infection. After 24 h incubation at 37 °C and 5% CO₂ atmosphere, the cells were harvested, purified, and stained with 200 nM MitoRed (Dojindo Molecular Technologies Inc. Japan) by following the manufacturer’s protocol. Fluorescence measurement was acquired by using a spectrofluorometer (Corona Electric, Japan) with excitation set at 560 nm and emission at 580 nm.

Measurement of intracellular ROS was as previously described (Adeyemi et al. 2017 (link)). This is premised on the intracellular peroxide-dependent oxidation of 2′,7′-dichlorodihydro-fluorescein diacetate (H₂DCF-DA, Sigma, St. Louis, MO, USA) to form the fluorescent compound 2′,7′-dichlorofluorescein (DCF). Briefly, growing HFF monolayers were treated with C. gigantea in the absence/presence of T. gondii infection. After 24 h incubation at 37 °C and 5% CO₂ atmosphere, the cells were harvested, purified, and re-suspended in PBS containing the H₂DCF-DA to a final concentration of 100 µM. The cell suspension containing the fluorescent dye was incubated for 30–60 min at 37 °C. Fluorescence acquisition was recorded by using a spectrofluorometer (Corona Electric, Japan) with excitation set at 485 nm and emission at 530 nm. H₂O₂ was included as a positive control in order to validate the ROS detection assay.