The loss of TAFAZZIN does not affect the production of neutrophil reactive oxygen species. (A) Schematic outlining commonly understood ROS pathways as well as reagents used to quantify and distinguish the different species within neutrophils. Microbial pathogens can be recognized and engulfed by neutrophils. In the process of phagocytosis, neutrophils activate membrane-associated NADPH oxidase to generate a powerful oxidative burse (measured by the Seahorse assay). During this oxidative burst, oxygen is consumed generating ROS including superoxide (O₂⁻) (measured by the Cytochrome C assay) and hydrogen peroxide (H₂O₂) (measured by the H₂DCF assay). The mitochondria are also an important source of ROS, in particular, O₂⁻ (measured by the MitoSOX reagent). (B) The oxygen consumption rate (OCR) was measured within the XF Neutrophil Activation Assay. Cells were stimulated with PMA, 100 ng/mL and OCR monitored over 240 minutes. This experiment was performed twice. (C) WT and TAFAZZIN-KO neutrophils were stimulated with PMA, fMLP, or RGD and the production of superoxide monitored over 60 minutes. The total superoxide (area under the curve) is displayed. This experiment was performed 3 times. (D) WT and TAFAZZIN-KO neutrophils were stimulated with PMA, fMLP, or RGD; the conversion of H₂DCF to its fluorescent product was quantified by flow cytometry and displayed as mean fluorescence intensity (MFI). Unstimulated cells are displayed as a negative control. This experiment was performed 3 times. (E) Mitochondrial ROS was quantified by MitoSOX staining and flow cytometry in both GMPs and mature neutrophils. Data are displayed the MFI normalized to the WT clones. This experiment was performed twice (GMP) and once (neutrophils). Error bars indicate the mean ± SD. t tests were performed. NS, not statistically significant.