Mitored

The measurement of mitochondrial membrane potential was acquired by spectrofluorimetry according to a procedure described by Baracca et al.26 (link) Briefly, growing HFF monolayers were infected with purified parasite suspension and incubated for 24 h at 37°C. The parasite-infested HFF monolayers were treated with the NPs and further incubated for 8 h at 37°C. The parasites were then harvested, purified, and stained with 200 nM MitoRed (Dojindo Molecular Technologies Inc, Kumamoto, Japan) by following the manufacturer’s protocol. Fluorescence acquisition was assessed using a spectrofluorometer with excitation set at 560 nm and emission at 580 nm.

Cells were seeded on glass cover slips and incubated with or without 100 µM AZT for 48 h. Mitochondria were then stained with 100 nM MitoRed (Dojindo Laboratories, Kumamoto, Japan) at 37 °C for 30 min and fixed with 4% formalin in PBS for 15 min at room temperature. To block non-specific binding of antibodies, the specimens were treated with blocking-permeabilization buffer containing 1% bovine
serum albumin fraction V (Sigma-Aldrich, St. Louis, MO, USA), 5% normal goat serum (Vector Laboratories, Burlingame, CA. USA), and 0.3% Triton X-100 (Sigma-Aldrich) in PBS for 1 h, followed by rabbit anti-Drp1 antibody (Cell Signaling Technology; 1:50 dilution in blocking permeabilization buffer) at 4 °C for 18 h. Samples were washed three times with PBS and then incubated with CF488A-conjugated goat anti-rabbit immunoglobulin G antibody (Biotium, Fremont, CA, USA; 1:500 dilution in PBS). Cell nuclei were stained with DAPI. Samples were mounted with Prolong Diamond Antifade Mountant (Molecular Probes, Eugene, OR, USA). Images of mitochondria were acquired to determine Drp1 localization using a structured illumination microscope with 100× CFI-SR Apochromat TIRF objective lens (NA = 1.49) (Nikon, Tokyo, Japan). Drp1 localization in mitochondria was evaluated by calculating the Pearson correlation coefficient using NIS Elements software (Nikon).

The MMP measurement was as described previously (Adeyemi et al., 2017a ). In brief, cultures of HFF cells treated with C. sinensis oil in the absence/presence of T. gondii infection were incubated for 24 h at an atmosphere of 37 °C and 5% CO₂. Thereafter, the cells were harvested, washed, and stained with 200 nM MitoRed (Dojindo Molecular Technologies Inc. Japan). Fluorescence was recorded on a spectrofluorometer with excitation set at 560 nm and emission at 580 nm.