Figure 5: Assessment of iNOS and NETs in ICAM-1⁺ neutrophils in vitro.

Purified BMDN (1 × 10⁶ cells) were stimulated with 1 µg/ml of rmCIRP for 4 h. Surface ICAM-1 was stained with APC-Ly6G Ab and FITC-ICAM-1 Ab, followed by fixing and permeabilization steps, intracellular staining for iNOS expression was performed by using PE-anti-iNOS Ab. (A, B) Representative dot blots and corresponding bar diagram are shown. Data are expressed as means ± SE (n = 6 mice/group) and compared by Student’s t test (*p < 0.05 vs. ICAM-1⁻). In order to assess NETs in vitro, a total of 1 × 10⁶ BMDN were stimulated with rmCIRP (1 µg/ml) at 37°C for 4 h. After fixing and blocking and without permeabilization of the cells, they were then stained with APC-Ly6G, BV421-ICAM-1 and FITC-MPO Abs. (C, D) Dot blots and corresponding bar diagram representing the frequencies of extracellular MPO in ICAM-1 expressing neutrophils are shown. Data are expressed as means ± SE (n = 4 mice/group) and compared by Student’s t test (*p < 0.05 vs. ICAM-1⁻). CIRP, cold-inducible RNA-binding protein; ICAM-1, intercellular adhesion molecule; rmCIRP, recombinant murine CIRP; BMDN, bone marrow-derived neutrophils.