Intracellular ROS Measurement in Leishmania

Intracellular ROS was measured using dichlorofluorescein diacetate (DCFDA) as previously described but with modifications [26 (link)]. Mid-log phase promastigotes were incubated for eight hours with or without 200 μM potassium antimonyl tartrate (Sigma-Aldrich). Parasites were then precipitated by centrifugation, washed once with PBS then resuspended in HEPES-NaCl assay buffer (21 mM HEPES, 137 mM NaCl, 5 mM KCl, 0.7 mM Na₂HPO₄, 6 mM glucose at pH 7.4) to a density of 7.5 x 10⁷ cells/ml with 4 μM DCFDA (Sigma-Aldrich). Two 200 μl aliquots taken from each sample were incubated for 40 min at 25 °C before measuring fluorescence on a FLUOstar Optima microplate reader (BMG Labtech) with excitation wavelength of 485 nm and emission wavelength of 520 nm. The average of these technical replicates was calculated, from which was subtracted a background value, determined as the average measurements of five wells containing only HEPES-NaCl buffer with 4 μM DCFDA.