Quantifying Oxidative Stress in Dictyostelium

The production of ROS was measured using DCFDA (2’-7’dichlorofluorescin diacetate) dye (1μg/ml, Sigma). Control and treated D. discoideum amoebae (1×10⁶ cells/ml) were starved in 1X KK2 buffer in the presence of thymoquinone for six hours, then gently washed with PBS followed by the addition of DCFDA, and incubated for 30 min at 22°C with gentle shaking. DAPI was added to aggregated cells and fluorescence was captured using a Nikon ES400 microscope [28 (link)]. Quantitation of DCFDA staining was performed by measuring intensity values of comparable sized clusters of aggregated cells for three independent samples and performing statistical evaluation of relative intensity using averages of five clusters/group, followed by the Student’s t-test.