Quantifying Mitochondrial Morphology in T. brucei

Live T. brucei cells (5 × 10⁶) expressing TbTim17 mutants were used for MitoTracker staining as previously described (69 (link)). Briefly, cells were washed twice with PBS and spread evenly over gelatin-coated slides. Once the cells had settled, the slides were washed with cold PBS to remove any unattached cells. The attached cells were fixed with 3.7% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking with 5% non-fat milk for 30 min, the slides were washed with 1× PBS. DNA was stained with 1 µg/mL 4′,6-diamidino-2-phenylindole. Cells were imaged using a Nikon TE2000E widefield microscope equipped with a 60 × 1.4 NA Plan Apo VC oil immersion objective. Images were captured using a CoolSNAP HQ2 cooled CCD camera and Nikon Elements Advanced Research Software. There were no max projections of a Z-stack. The software used to calculate the Pearson’s coefficient is Nikon NIS Elements Advanced Research Imaging Software.