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. Author manuscript; available in PMC: 2022 Feb 15.
Published in final edited form as: J Immunol. 2020 Dec 30;206(4):797–806. doi: 10.4049/jimmunol.2000091

Figure 1: eCIRP-induced NETs inhibit efferocytosis.

Figure 1:

(A, B) A total of 5 × 105 peritoneal macrophages were cultured with 1.5 × 106 CFSE-labeled apoptotic cells in presence with PBS or various concentrations of NETs. After 1 h of incubation, cells were washed, fixed with 2% PFA, and efferocytosis was assessed by flow cytometry. Efferocytosis was determined as the percentage of CFSE-positive cells present in F4/80+ macrophages. Data were obtained from 3 independent experiments and expressed as means ± SE (n=7 samples/group). The groups were compared by one-way ANOVA and SNK method (*p<0.05; ***p<0.001 vs. PBS-treated group). (C) Confirmation of phagocytosis of apoptotic cells in F4/80 and CFSE double-positive population by image stream analysis. Representative images showing co-localization of F4/80 and CFSE double positive cells indicate efferocytosis. Scale bar: 7 μm. BMDN, bone marrow-derived neutrophil(s); MPO, myeloperoxidase; CFSE, carboxyfluorescein succinimidyl ester; PFA, paraformaldehyde; NETs, neutrophil extracellular traps; rmCIRP, recombinant mouse cold-inducible RNA-binding protein.