Fitc rat anti mouse cd45

Flow cytometry was performed as previously reported (7 (link)). Briefly, after perfusion of the kidneys with PBS, 1 kidney was removed, minced into fragments, and digested in RPMI 1640 containing 2 mg/ml collagenase type D and 100 μg/ml DNase I for 45 minutes at 37°C, with intermittent agitation. Kidney fragments were passed through a 40 μm mesh (Falcon; BD Biosciences), yielding single-cell suspensions. Cells were centrifuged (300g, 10 minutes, 4°C), resuspended in FACS buffer, kept on ice, and counted using a Bio-Rad TC20 automated cell counter. 10⁵ cells were incubated in 2.5 μg/ml Fc blocking solution and stained for 60 minutes at 4°C with antibodies, including FITC rat anti-mouse CD45, APC anti-Ly6G, PE/Cy7 anti-mouse F4/80, Pacific Blue anti-mouse CD11b, APC anti-mouse CD11c, or isotype control (all BioLegend). Cells were then washed and stained for 15 minutes at room temperature with annexin V or H₂DCFDA and resuspended in 1× annexin binding buffer (or PBS with 1% BSA for H₂DCFDA). For cell cycle analysis, cells were stained with propidium iodide at 50 μg/ml. After immunostaining, cells were analyzed immediately on a Novocyte flow cytometer with NovoExpress Software (Acea Biosciences) for data acquisition, and data analysis was performed using FlowJo v10 software (Tree Star).

The kits for alanine aminotransferase (ALT), aspartate aminotransferase (AST), and triglyceride (TG) were supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α and IL-1β were purchased from Bender MedSystems (Vienna, Austria). PE rat anti-mouse Ly6G was obtained from BD Biosciences (New Jersey, USA). Neutrophils and F4/80 antibodies were obtained from Thermo Scientific (Rockford, IL, USA). Rabbit anti-mouse TLR4 antibody, rabbit anti-mouse phospho-IRAK1, phospho-p38, phosphor-IKKβ, IKKβ, phospho-IκBα, IκBα, phospho-p65, p38, p65, β-actin, Lamin B1, and GAPDH were purchased from Abcam (Cambridge, UK). PE rat anti-mouse F4/80, FITC rat anti-mouse CD11b, and FITC rat anti-mouse CD45 antibodies were from Biolegend, Inc. (San Diego, USA). TLR4-specific antagonist TAK-242 was from MedChemExpress LLC (Shanghai, China). Palmitic acid (PA) was from Sigma-Aldrich (St. Louis, USA).