Assessing Auranofin's Impact on N. fowleri Antioxidants

To assess the effect of auranofin on antioxidant pathways of N. fowleri, 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) (Sigma) was used. DCFDA is a cell-permeable molecule which is oxidized by the reactive oxygen species inside the cell forming the fluorescent dichlorofluorescein (DCF) ^{64 (link)}. Reactive oxygen species production assay was optimized for N. fowleri following earlier method ^{24 (link)}. Briefly, 100 μL (20,000 amebae) of N. fowleri KUL trophozoites were preincubated in a transparent, flat bottom 96-well plate (Fisher Scientific) with 0.5% DMSO, 3 μM auranofin in duplicate for 18 h. In another set, compound-treated and 0.5% DMSO-treated trophozoites were incubated with 300 μM H₂O₂ for 2 h. After 2 h, media were removed from the wells, washed and replaced with prewarmed Nelson’s medium containing 0.4 mM of DCFDA. The plate was incubated at 37°C for 30 min in the dark. Each well was washed again and pictures of the fluorescent cells were taken with an Axio Vert.A1 inverted phase microscope (Zeiss) and Zen lite software (Zeiss).