Brilliant violet 421 anti mouse f4 80 antibody

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).

After carefully removing the muscle tissue and ligament around the spine, separate and cut the endplates with a surgical blade under a stereomicroscope. Flush the endplates with fluorescence‐activated cell sorting (FACS) buffer (5% FBS in PBS), and cut the tissue into 1 mm³ fragments with sterile scissors. The endplates were further digested with 0.25% trypsin (Solarbio) for 20 min and 0.25% Collagenase $\mathrm{II}$ (Solarbio) solution for 1.5 h at 37 °C and passed through a 40 µm cell strainer to yield single‐cell suspensions. The cells were collected and resuspended in red blood cell lysis buffer (Solarbio) for the lysis of red blood cells. After washing, cells were resuspended in 100 µl FACS buffer and incubated with Brilliant Violet 421™ anti-mouse F4/80 antibody (1:100, 123131, Biolegend, Inc., San Diego, CA) for 30 min at 4 °C. After washing, cells were analyzed using a BD LSRFortessa flow cytometer. The data were analyzed with FlowJo software (version 10, BD Bioscience).