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. 2022 Oct 5;8(40):eabq5384. doi: 10.1126/sciadv.abq5384

Fig. 3. Functional alteration of neutrophils in serum iron deficiency.

Fig. 3.

(A) Phenotyping ex vivo of isolated BM neutrophils (obtained from mice treated as in Fig. 2A). Dihydrorhodamine 123 (DHR123) fluorescence as a reporter for cytoplasmic ROS in PMA-stimulated cells. Phagocytosis of GFP-labeled E. coli. FMO, fluorescence minus one. Two-way ANOVA. Mean, quartiles, and range. Bacterial killing of S. aureus, resolved as colony-forming units of S. aureus recovered after coculture with neutrophils. Unpaired t test. Means ± SD. (B) Supernatant CCL2 and TNF levels produced by zymosan-stimulated neutrophils measured by enzyme-linked immunosorbent assay (ELISA). Unpaired t test. Mean, quartiles, and range. (C) NETosis was evaluated in isolated BM neutrophils after stimulation ex vivo with PMA and ionomycin ± DPI. A minimum of 250 cells from two replicates per sample counted. Two-way ANOVA. Mean, quartiles, and range. Representative microscopy images taken at ×20 magnification. (D) NETosis was evaluated in isolated BM neutrophils after stimulation ex vivo with PMA ± apocynin (APO). A minimum of 250 cells from two replicates per sample counted. Two-way ANOVA. Mean, quartiles, and range. Representative microscopy images taken at ×20 magnification. (E) SCENITH (single-cell metabolism by profiling translation inhibition) analysis of Ly6G+ BM neutrophils from experiment described in Fig. 1A, analyzing proportional shifts in O-propargyl-puromycin (OPP) incorporation after ex vivo treatment with metabolic inhibitors as detailed in Materials and Methods. Two-way ANOVA with matching for sample. Reported P value is the effect of mHep treatment. Mean. FAO, fatty acid oxidation; AAO,amino acid oxidation. (F) MitoSOX fluorescence as a reporter for mitoROS in ionomycin stimulated isolated BM neutrophils. Two-way ANOVA. Mean, quartiles, and range. (G) NETosis was evaluated in isolated BM neutrophils after stimulation ex vivo with ionomycin (iono) ± mitoTEMPO. A minimum of 250 cells from two replicates per sample counted. Two-way ANOVA. Mean, quartiles, and range. Representative microscopy images taken at ×20 magnification.