TAFAZZIN deficiency results in cardiolipin changes but does not affect neutrophil function. (A) Quantification of cardiolipin and monolysocardiolipin content in WT and TAFAZZIN-KO GMPs. Total CL and monolysocardiolipin values are normalized to the average of WT cells. (B) The degree of unsaturation within the cardiolipin acyl chains was compared between WT and TAFAZZIN-KO cells. The average of the 4 clones is displayed, demonstrating the expected decrease in number of unsaturations in the TAFAZZIN-KO cells. (C) Imaging flow cytometry was used to confirm phagocytosis of FITC-labeled BioParticles within the WT or TAFAZZIN-KO mature neutrophils. The intracellular S aureus or E coli BioParticles (green) can be quantified separated from the extracellular particles simply adherent to the cell’s surface. CD11b (blue) was used to highlight the membrane and 4′,6-diamidino-2-phenylindole (red) the nuclei of the neutrophils. (D) The degree of in vitro phagocytosis was quantified by flow cytometry across multiple WT and TAFAZZIN-KO clones. This assay was performed on 3 independent occasions. (E) In vivo analysis of phagocytosis was performed in a limited number of adult mice (CD45.1) that had been transplanted with WT or TAFAZZIN-KO fetal liver hematopoietic cells (CD45.2), as described in Figure 2A. Mice received an intraperitoneal injection of BioParticles and peritoneal fluid was collected at 3 hours for flow cytometric analysis. This experiment was performed twice and data from both experiments combined. (F) The amount of TNF-α, NGAL, and ELA2 secreted from LPS-stimulated (4 hours, 10 ng/mL) mature neutrophils was quantified by enzyme-linked immunosorbent assay. This experiment was performed twice. Error bars indicate the mean ± SD. t tests were performed. ***P < .001. NS, not statistically significant.