Figure 3.
ANCA-related neutrophil functions are similar in NGAL−/− and WT neutrophils. (A and B) Murine WT or NGAL−/− neutrophils were isolated from the BM and stimulated with buffer (Buf), anti-BSA IgG, anti-MPO IgG, PMA, or zymosan (Zym). (A) Intracellular ROS generation was measured by dihydrorhodamine 123 staining and flow cytometry. (B) Extracellular ROS generation was detected by ferricytochrome reduction assay. (C) Neutrophils were incubated overnight in culture medium and spontaneous apoptosis was measured using Annexin V staining and flow cytometry. (D) Neutrophils were incubated with FITC-labeled particles and phagocytosis (FITC+ neutrophils) was analyzed by flow cytometry. (E) Chemotactic migration toward colony-stimulating factor 2 (CSF2, 50 ng/ml) and CXCL1 (400 ng/ml) was assessed. (F) Neutrophils were stimulated with either TNF or TNF/anti-MPO IgG and NET generation was detected with Sytox green staining. Representative images of three independent experiments are depicted at low magnification (20×). (G) Degranulation was measured by PR3 FRET assay in culture supernatants of WT and NGAL−/− neutrophils stimulated with TNF/anti-MPO IgG. Results are reported as Vmax. Buffer and anti-BSA IgG were used as controls. MFI, mean fluorescence intensity; O2−, superoxide. ***P<0.001.