Anti il 1r

Lipopolysaccharide (LPS, 1.5 mg/kg) was injected intraperitoneally into the mice to activate neutrophils in vivo. Neutrophils were collected from bone marrow of mice after 6 h post injection, by Percoll gradient method described previously [34 (link)]. Neutrophils (10⁷) were cultured in a 100-mm dish for 12 h in RPMI 1640 media and 10 mL supernatant were collected. Then neutrophils-derived exosomes were isolated from the harvested supernatant by using a supercentrifuge rotor. Briefly, the supernatant from neutrophils were centrifuged at 400 g for 10 min, 1250 g for 15 min, and 10,000 g for half an hour at 4 °C to remove cells and debris. After filtered using a 0.22-mm filter (Millipore, Billerica, MA, USA), the solution was centrifuged at 100,000 g for 70 min at 4 °C using an ultracentrifuge (Beckman Coulter, Brea, CA, USA). The exosome pellet was collected and resuspended in PBS, and ultracentrifuged again at 140,000 g for 70 min. Finally, the pelleted neutrophil-derived exosomes were obtained, and the protein quantification were determined by using a Micro BCA Protein Assay kit. To detect neutrophil marker (LFA-1, anti-IL-1R, anti-TNF-αR) and exosome markers (CD9, CD63 and TSG 101), western blotting was carried out with anti-LFA, anti-IL-1R, anti-TNF-αR, anti-CD9, anti-CD63 and anti-TSG 101 antibodies (Abcam, Cambridge, UK).

After being denatured by heating at 95°C in an SDS sample buffer, the
supernatant samples were loaded onto Mini-Protean TGX Precast gels (Bio-Rad
Laboratories, Hercules, CA, USA) and electrically transferred to a polyvinylidene
fluoride membrane. The membrane was then incubated overnight with primary antibodies:
rabbit anti-IL-1R, anti-IL-6R and anti-TNFR1 (Abcam Co., Cambridge, UK, diluted
1:200). After being fully washed, the membrane was incubated with horseradish
peroxidase-linked anti-rabbit secondary antibody and visualized for immunoreactivity.
The membrane was stripped and incubated with mouse anti-β-actin to show equal
loading of the protein. The bands recognized by the primary antibody were visualized
by exposure of the membrane onto an X-ray film. Then, the film was scanned and the
optical densities of IL-1R, IL-6R, TNFR1 and β-actin bands were determined
using the public domain ImageJ program (made by Wayne Rasband, Scion Corporation,
Bethesda, MD, USA). Then, values for densities of immunoreactive bands/β-actin
band from the same lane were determined. Each of the values was then normalized to a
control sample.