Anti cxcr2 apc

Expression of cell surface markers was measured after blocking of unspecific binding sites with CD16/CD32Fc-Block (BD Biosciences, San Jose, CA, USA) by staining cells with anti-Gr-1-APC (BD Biosciences, San Jose, CA, USA, Clone RB6-8C5), anti-Ly6G (Gr-1)-PE (eBioscience, Clone RB6-8C5) anti-CD11b-PE (AbD serotec, Bio-Rad, München, Germany, Clone M1/70), anti-c-kit-APC (eBioscience, San Diego, CA, USA, Clone ACK2), anti-CXCR2-APC (Biolegend, San Diego, CA, USA, Clone TG11/CXCR2) or anti-CXCR4-APC (BD Biosciences, Heidelberg, Germany, Clone 2B11/CXCR4) followed by flow cytometry analysis on a FACS Calibur or Fortessa (BD Biosciences).

Dissected mouse livers were minced with a scalpel on ice and incubated at 37 ˚C in RPMI-1640 media containing 0.05% collagenase/dispase (11097113001; Roche) and 0.01% trypsin inhibitor (T7659; Sigma). Cell suspension was filtered through a 70 μm cell strainer into PBS and residual red blood cells lysed using red cell lysis buffer (11814389001; Roche). Cell suspensions at a concentration of 1 × 10⁷ cells/ml were the incubated for 45 min at 4 ˚C in the presence of anti-CD11b PE-Cy7 (25-0112-82, eBioscience), anti-Gr1 PE (12-5931-82; eBioscience), anti-CD45 PE (12-0451-82; eBioscience), anti-CXCR2-APC (149305; Biolegend), and Mouse BD Fc Block (553141, BD Bioscience). FACS analysis was performed using a FACSCalibur flow cytometer (BD Biosciences) and analyzed with FlowJo software version 7.2.5 (Tree Star, Ashland, OR).

Staining for cellular and nuclear morphology was performed on cytospins from cultures of progenitors or differentiated neutrophils by incubating with Giemsa solution (Merck, Darmstadt, Germany) after methanol fixation. Analysis by brightfield microscopy was performed using a Keyence BZ9000 microscope at a magnification of x40 (Keyence, Neu-Isenburg, Germany).
Expression of cell surface markers was measured by staining cells with anti-Gr-1-FITC (BD Biosciences, San Jose, CA, USA), anti-CD11b-APC (eBioscience, San Diego, CA, USA), anti-c-kit-APC (eBioscience) or anti-CXCR2-APC (Biolegend, San Diego, CA, USA) followed by flow cytometry analysis on a FACS Calibur (BD Biosciences, Heidelberg, Germany).