Immunoblot and Flow Cytometry for Neutrophil Characterization

For immunoblot analysis, S087 rabbit immune serum was used to detect both Rab27a and Rab27b. Slp1 and Munc13-4 rabbit immune serum used for immunoblot were kindly provided by Dr. Sergo D. Catz (The Scripps Research Institute, La Jolla, CA, USA) and were described previously [17 (link),37 (link)]. Immunoblots were performed as described previously [14 (link)]. For flow cytometry using a FACScalibur cytometer, phycoerythrin (PE)-conjugated anti-Ly6G (BD Biosciences) and allophycocyanin (APC)-conjugated CXCR2 (R&D Systems) antibodies were used at 1:100 dilution. For surface antigen staining, 2 × 10⁵ bone marrow derived neutrophil (BMDN) cells were washed twice and resuspended in rat anti-FcγRII/RIII antibody (BD biosciences) for 10 minutes, then incubated with fluorophore-conjugated antibodies for 30 minutes on ice.