Pe conjugated anti mouse human cd11b antibody

For flow cytometry analysis, the cells were washed twice with PBS, and 100-µl aliquots of the cell suspensions were protected from light and incubated with 10 µl of a PE-conjugated mouse anti-human CD11b antibody (BioLegend, San Diego, CA) for 60 min at room temperature. Isotype-matched PE-conjugated mouse IgG (BioLegend) antibody was used as a negative control. After the incubation, the samples were applied to a MACSQuant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). The mean fluorescence intensity (MFI) was calculated by subtracting the value of the mean fluorescence channel of the respective isotype control from the value obtained from the sample incubated with the specific antibody.

For FCM analysis, approximately 2–3×10⁵ cells were washed with PBS, and 30 μl aliquots of the cell suspensions were protected from light and incubated with 1 μl of a phycoerythrin (PE)-conjugated mouse anti-human CD11b antibody (BioLegend, San Diego, CA) for 30 min at room temperature. An isotype-matched PE-conjugated mouse IgG (BioLegend) antibody was used as a negative control. After incubation, the samples were analyzed on an LSR Fortessa X-20 Flow cytometer (BD Biosciences, San Jose, CA).

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.