Analyzing Trypanosome Respiration with MitoTam

The effect of MitoTam on respiration was analyzed by Oroboros Oxygraph-2K (Oroboros Instruments Corp., Innsbruck, Austria) as described (59 (link)). Bloodstream form T. brucei cells were incubated with 40 nM or 100 nM MitoTam for 16 h and 24 h, as indicated. For each treated sample and control, 2 × 10⁷ cells were spun down (1,400 g, 10 min, RT) and pellets were washed in Mir05 medium (0.5 mM EGTA, 3 mM MgCl₂, 60 mM lactobionic acid, 20 mM taurine, 10 mM KH₂PO₄, 20 mM HEPES, 110 mM sucrose, 1 mg/mL fatty acid-free BSA, pH 7.1). Before the measurement started, the pellets were resuspended in 0.5 mL of Mir05 medium preheated to 37°C and transferred to the respiration chamber. Respiration was monitored at 37°C and with constant stirring. The experiment started with the addition of 10 mM glycerol-3-phosphate (Sigma), the mitochondrial glycerol-3-phosphate dehydrogenase substrate, and respiration was inhibited by the addition of 250 μM SHAM (Salicylhydroxamic acid), the inhibitor of the trypanosomal alternative oxidase. The acquired data were analyzed using Prism (8.0) (GraphPad Software).