Mitochondrial Staining in Parasites

To stain mitochondria, live parasites were resuspended in DMEM at 1.0 × 10⁶ cells/ml and labeled with 100 nM of Mitotracker CMXRos or TMRE. After incubation at 27 °C (30 min for Mitotracker and 15 min for TRME), parasites were washed once with PBS, fixed and processed for fluorescence microscopy as previously described [15 (link)]. MitoSox Red labeling (5 μM for 25 min at 27 °C) of live parasites was performed similarly without fixation using an Olympus BX51 Upright Fluorescence Microscope. DNA was stained with DAPI (2.5 μg/ml). Transmission electron microscopy of log and stationary phase promastigotes was performed as previously described [76 (link)].