Assessing Mitochondrial Membrane Potential in Parasites and Host Cells

Extracellular parasites (1 × 10⁵ per group) collected as described in Section “Parasites” were maintained in suspension with myrislignan (32 or 70 μg/ml) in DMEM or without any drug (positive or negative control) for 8 h at 37°C. The negative control samples were incubated for 20 min with 10 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which can cause ΔΨm depletion. After incubation, the tachyzoite samples were stained with the MitoTracker Red CMXRos probe (50 nM, Invitrogen, USA) for 20 min, as described previously with some modifications (Besteiro et al., 2011 (link)).
Vero cells were placed in cell culture dishes and incubated with myrislignan (70 μg/ml) or without any drug (as a positive control) in DMEM for 8 h at 37°C, stained with the MitoTracker Red CMXRos probe (250 nM) for 20 min, rinsed twice with PBS, fixed with 4% polyformaldehyde for 15 min, and washed twice with PBS to remove the polyformaldehyde, stained with Hoechst 33342 (C1027, Beyotime Institute of Biotechnology, China). The fluorescence intensity was observed by laser confocal microscopy. The relative fluorescence intensity of the cells was measured using an Enspire Microplate Reader (PerkinElmer, German).