Anti cd66b pe cy7

Peripheral blood mononuclear cells (PBMCs) were isolated via centrifugation on a Ficoll–Paque PLUS gradient (GE Healthcare, Uppsala, Sweden). Neutrophils were further isolated after dextran sedimentation and hypotonic lysis as previously described (20 (link)).
Fresh EDTA anti-coagulated peripheral blood (100 μl) was incubated with antibodies for 30 min in the dark. The following antibodies were used: Anti-CD66b-PE-cy7, anti-TLR2-FITC, anti-TLR4-APC, anti-CXCR1-APC, anti-CXCR2-FITC, anti-C5aR-APC, anti-CD64-PE, anti-CD177-APC, anti-CD62L-FITC, anti-CD11b-PE, anti-CD49d- FITC, anti-C5L2-PE, anti-CD3-BV421 and anti-PD-1-PE were obtained from Biolegend (San Diego, California, USA); anti-CD4-BV421, anti-CD8-APC-cy7, anti-CD38-FITC, and anti-HLA-DR-PerCP were purchased from BD Biosciences (Franklin Lakes, New Jersey, USA); anti-PD-L1-PE were bought from eBioscience (San Diego, California, USA). Isotype-matching antibodies were used as negative controls. After lysing the red blood cells, the remaining cells were washed and fixed for flow cytometry analysis.

Cells were washed twice in phosphate-buffered saline (PBS) with 1% bovine serum albumin and 1% mouse serum and then stained with a mixture of antibodies against surface antigens: anti-CD66b (PE/Cy7-labeled, Biolegend #305115) at 1:50, anti-CD16 (PerCP-labeled, clone 3G8 Biolegend #302029) at 1:200, anti-CD14 (PE-labeled anti-human CD14 antibody clone 63D3 (Biolegend #367103) at 1:200, and anti-CD19 (APC/Cyanine7-conjugated, clone HIB19, Biolegend #302218) at 1:200, anti-CD15 (PerCP-conjugated, clone W63D, Biolegend #323018) at 1:200 in PBS with 1% BSA and 1% mouse serum for 30 min at 4 °C in the dark. The cells were washed twice, fixed in 1% paraformaldehyde in PBS, permeabilized in 200 μl 0.1% Tween in PBS for 30 min at room temperature, washed and resuspended in 50 μl the same buffer with 1:500 diluted anti-L1 p40 (clone 4H1, EMD Millipore MABC1152) conjugated to APC using Mix-n-Stain™ Fluorescent Protein & Tandem Dye Antibody Labeling Kits (Biotium #92306). After 30 min at 4 °C in the dark, the cells were washed twice with PBS, 1% BSA, and 1% mouse serum and resuspended in 200 μl of this buffer for analysis on a CytoFLEX Flow Cytometer (Beckman Coulter).