Fitc anti mouse ly 6g antibody

Mice were treated with testosterone (i.p. 10 mg/kg/day) or flutamide (s.c 7 mg/kg/day; Sigma–Aldrich, St. Louis, MO) for 3 days. Then, neutrophil recruitment was induced by a single i.p. injection of LPS from E. coli O111:B4 (0.5 mg/kg, Sigma–Aldrich, St. Louis, MO). Six hours after, mice received a single i.v. dose of a FITC anti-mouse Ly-6G antibody (4 μg/mouse; BioLegend, San Diego, CA), diluted in sterile saline (in a total volume of 100 μl), and confocal intravital imaging was performed as described (23 (link), 24 (link)). In brief, mice were anesthetized (i.p.) with a mixture of ketamine (60 mg/kg) and xylazine (15 mg/kg) and a midline laparotomy was performed to expose the liver for imaging. Mice were imaged using Nikon Eclipse Ti (Nikon, Tokyo, Japan) with a C2 confocal head equipped with three different lasers (excitation at three wavelengths: 405, 488, and 543 nm) and emission bandpass filters at 450/50, 515/30, and 584/50 nm. The z-position is controlled by an automated device and 10X objective was used on the required resolution. Ten-minute movies were taken from each mouse and Ly-6G (+) neutrophil quantification was performed using Volocity 6.3 software (PerkinElmer, Waltham, MA).

LPS and nigericin were purchased from Sigma-Aldrich (St. Louis, MO). NF-kB inhibitor, QNZ (EVP4593), was supplied by MedChemExpress (New Jersey, USA). Antibodies against FTO, NLRP3, FoxO1, P65, p-P65, IL-1β and Cleaved-IL-1β (Asp117) were obtained from Cell Signaling Technologies (Beverly, MA). ELISA kits of IL-1β, interleukin-6 (IL-6), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10) and interleukin-12(p70) (IL-12(p70)) were purchased from eBioscience (San Diego, CA). Brilliant Violet 421™ anti-mouse F4/80 antibody, PE anti-mouse/human CD11b antibody, FITC anti-mouse I-A/I-E antibody, APC anti-mouse CD80 antibody, PE/Cy7 anti-mouse CD86 antibody, FITC anti-mouse Ly-6G antibody and APC anti-mouse CD40 antibody were obtained from BioLegend (San Diego, CA, USA). Lipidoid (C12-200) was supplied by Xinjiahecheng Medical Chemistry Corporation (Wuhan, Hubei, China). mPEG2000-DEG was purchased from NOF Corporation (Tokyo, Japan).

Mice were sacrificed 24 h after LPS exposure. Lung tissues and peripheral blood mononuclear cells were collected. To obtain BAL fluid, lungs were washed three times with 1 ml PBS. The single-cell suspensions for flow cytometric analysis were pre-incubated with anti-CD16/CD32 antibody (Cat# 101301, from BioLegend, San Diego, CA, USA) to block Fc receptors, and then stained with APC anti-mouse CD45 antibody (Cat# 103137, from BioLegend) and FITC anti-mouse Ly6G antibody (Cat# 127605, from BioLegend) at 4 °C for 20 min. Then, total cell numbers of immune cells including neutrophils were determined through the Cytoflex Flow Cytometer (Beckman Coulter, CA, USA).