α_Mβ₂ is present on the surface of PMN-derived MPs. (A,B) Human PMNs were resting or stimulated with PMA (50 nM), PAF (500 nM), or LPS (5 μg/mL) in HBSS buffer containing Ca²⁺/Mg²⁺ ions (1 mM each) and PE-conjugated anti-α_M mAb (clone ICRF44, black, open histograms) or PE-labeled mouse isotype control (IgG₁) antibody (gray-filled histograms) for 30 minutes at 37°C. Samples were fixed with 1% paraformaldehyde and analyzed by FACS. The histogram and graph data are mean fluorescent intensities (MFIs) (± SEM) of 10 000 events from gate R2 representing MPs (Figure 1A dot plot). The data are representative of 5 experiments performed with PMNs from 5 different blood donors. (C) Protein concentration in samples of MPs derived from resting or PMA-stimulated PMNs was determined, and equal amounts of protein were resolved on 9% (left) or 6% (right) sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions and immunoblotted with mouse anti–human α_M or PSGL-1 to confirm equal loading of resting and PMA MPs. The band patterns are representative of 3 Western blots from 3 different MPs preparations.