Comparative Trypanosomatid Growth Inhibition

L. major promastigotes (Friedlin strain; WHO designation MHOM/JL/81/Friedlin), procyclic trypomastigotes of T. brucei brucei S427 29-13 and epimastigotes of T. cruzi CL Brener (genome project standard clone) were adapted for growth in SDM-79 medium supplemented with 10% fetal bovine serum (Gibco, Paisley, UK) and haemin (100 mg·L⁻¹). L. major promastigotes were grown at 24 °C with shaking, and T. brucei and T. cruzi were cultured at 28 °C. T. brucei bloodstream forms were cultured at 37 °C in modified HMI9 medium (56 μm 1-thioglycerol was substituted for 200 μm 2-mercaptoethanol) supplemented with 2.5 μg·mL⁻¹ G418 to maintain expression of T7 RNA polymerase and the tetracycline repressor protein [34 (link)].
In order to directly compare the effects of methylglyoxal on the growth of these trypanosomatids, triplicate cultures containing methylglyoxal were seeded at 5 × 10⁵ parasites per mL. As methylglyoxal interferes with the Alamar blue assay for viable cells, cell densities were determined using the CASY Model TT cell counter (Schärfe, Renlingen, Germany) after culture for 72 h. Concentrations of inhibitor causing a 50% reduction in growth (EC₅₀) were determined using the following two-parameter equation by nonlinear regression using grafit: where the experimental data were corrected for background cell density and expressed as percentages of the uninhibited control cell density. In this equation, [I] represents inhibitor concentration, and m is the slope factor.