Evaluating Inhibitors of T. brucei

Flasks of T. brucei were seeded at 1 × 10⁴ cells ml⁻¹ and incubated in the presence of the known inhibitors pentamidine and eflornithine, and the LOPAC hit (+)-U50,488 for 3 days. Flasks were set up in triplicate for each inhibitor at multiples of EC₅₀ along with 3 control flasks with no inhibitor added. Pentamidine and (+)-U50,488 were added in DMSO in a volume of 0.1% of the culture. Eflornithine was dissolved directly in HMI9-T medium and sterilised by 0.22 μM filtration before diluting into the cell culture. Cell densities were determined at intervals using a haemocytometer and generation times calculated using GraFit (version 5.0.13; Erithacus software) using the following equation: $N=N_0{2}^{t/g}$ where N₀ and N are the number of cells at time zero and time t, respectively, and g is the time per generation. For low cell densities, samples were concentrated 150-fold by centrifugation and resuspension in an appropriate volume of medium.