α_Mβ₂ Present on PMN-derived MPs is in active conformation and functional. (A-C) The samples were prepared from human PMNs and analyzed as described in Figure 1. The mAbs used were directed to activation-dependent epitopes within the α_M subunit: CBRM1/5-PE (A), mAb 24-Alexa488 (B) (black, open histograms), or mouse antibody isotype controls (PE-IgG₁) and Alexa488-IgG₁ (gray-filled histograms). (C) Alternatively, the samples were stained in the same FACS tube with mixtures of CBRM1/5-Alexa488 plus ICRF44-PE or mAb24-Alexa488 plusICRF44-PE mAbs, and ratios of binding of the α_Mβ₂ activation-specific mAbs to total α_Mβ₂ (measured with ICRF44) were calculated. (D) Human PMNs were pretreated with function-blocking mAbs to the α_M (44a), the β₂ (IB4), or MHC-1 (W6/32) mAbs (10 μg/mL) for 15 minutes at 37°C and then either left resting or stimulated with PMA (50 nM) in the absence or presence of Alexa488-labeled human Fg (20 μg/mL) for 30 minutes at 37°C. The samples were fixed, and 10 000 events falling into gate R2 (Figure 1A dot plot) were analyzed. The data are MFI (± SEM) and are representative of 5 independent experiments.