Fig 7. Cytotoxicity evaluation of ethyl pyruvate on human erythrocytes.

(A) 6x10⁵ red blood cells were mixed to 2x10⁵ T. brucei cells in 5 ml fresh medium in a flask. Cells were first incubated at 37°C in 5% CO₂ for 24 hrs. Out of this 1 ml-aliquots were distributed into 24-well plates and incubated with 100 μl of increasing concentrations of ethyl pyruvate (1–15 mM). Controls contained cells without ethyl pyruvate but 100 μl of fresh medium. The cells were then re-incubated for 3 hrs. Afterwards, the number of trypanosomes and human erythrocytes was counted using a haemocytometer. (B) Effect of ethyl pyruvate (10 mM) on red blood cell haemolysis was determined by measuring the optical density of the medium containing the T. brucei and red blood cells co-incubated for 3 hrs. Before measurement, the cells were spun down by centrifugation. Total haemolysis of an equivalent number of cells (labelled as ‘Control’) was achieved by sonication (70% power, 5x5 sec) using an ultrasonicater. Cells without ethyl pyruvate were used as negative controls (labelled as ‘Blank’).