Fitc conjugated anti mouse ly6g antibody

LPS (Escherichia coli 0111:B4) was from Sigma-Aldrich. Dextran sulfate sodium (DSS, MW 47,000; MP Biomedicals) was purchased from MP Biomedicals. Antibodies against phospho-AKT473, phospho-ERK1/2, phosphor-STAT1, AKT, ERK1/2, STAT1, GAPDH were purchased from Cell Signaling Technology, PHLPP was from EMD Millipore, β-ACTIN was from Santa Cruz. FITC conjugated anti-mouse LY6G antibody, Alexa Fluor 647 conjugated anti-mouse CD182 (CXCR2) Antibody, APC/Cy7 and PE conjugated anti-mouse/human CD11B antibody were from Biolegend. ELISA Kit for TNFα, IL-1β, and IL-6 were from eBioscience, respectively. MPO ELISA Kit from R&D system. leukotriene B4 (LTB4), N-Formyl-Met-Leu-Phe (fMLF), and percoll were purchased from Sigma-Aldrich. EasySep™ Mouse Neutrophil Enrichment Kit was purchased from Stemcell Technologies. MIP-1α, fibronectin, and G-CSF was from Peprotech. Anti-PMP70 was from ThermoFisher Scientific. Anti-LAMP was from Abcam.

LPS (Escherichia coli 0111:B4) was obtained from Sigma Aldrich. TPM from 3R4F reference cigarettes was prepared as described previously and provided by R.J. Reynolds Tobacco Company, Winston-Salem, NC (22 (link)). FITC conjugated anti-mouse Ly6G antibody, PE/Cy7 conjugated anti-mouse TNFa antibody, APC/Cy7 and PE conjugated anti-mouse/human CD11b antibody, were from Biolegend (San Diego, CA).

Mice were euthanized 24 hours after CLP or sham-operated surgery to get blood. Cells were surface-stained with FITC-conjugated anti-mouse Ly6G antibody (127605, BioLegend, San Diego, California, USA), fixed, and permeabilized before intracellular staining with PE-conjugated anti-mouse GSDMD antibody (ab246713, Abcam, Massachusetts, USA) which pre-absorbed against cells overnight at 4°C. The stained cells were analyzed by flow cytometry operated on FACSCanto II flow cytometer (BD Bioscience, San Jose, CA, USA) and the data were analyzed using FlowJo V7.6 (Tree Star, Ashland, OR, USA).