Figure 4
Figure 4. SCIPs, but not spread platelets, induce neutrophil spreading under flow. Washed platelets (2.5 × 108/mL) in unsupplemented Tyrode buffer were allowed to form monolayers in glass microcapillary tubes for 30 minutes at 37°C followed by blocking with 3% HSA for 15 minutes. Monolayers were left untreated (Spread) or pretreated with 1 μM ionophore A23187 for 10 minutes followed by ionophore removal with cell-free buffer to convert spread platelets to SCIPs (SCIP). Isolated neutrophils (106/mL) were then perfused over the monolayers at 150 s−1 for 5 minutes. (A) Scanning electron microscopy images of spread platelet and SCIP monolayers prior to neutrophil perfusion. (B) Scanning electron microscopy images of neutrophils that have interacted with Spread platelet or SCIP monolayers. (C) Neutrophil-platelet interactions were visualized by phase-contrast microscopy and the proportion of adherent neutrophils exhibiting stationary (i) or spread (ii) morphology quantitated. Images in panels A and B are representative of more than 10 individual experiments. Bar is 5 μm. Results in C are expressed as mean (±SEM; n = 4). *P < .05, **P < .01, ***P < .001.

SCIPs, but not spread platelets, induce neutrophil spreading under flow. Washed platelets (2.5 × 108/mL) in unsupplemented Tyrode buffer were allowed to form monolayers in glass microcapillary tubes for 30 minutes at 37°C followed by blocking with 3% HSA for 15 minutes. Monolayers were left untreated (Spread) or pretreated with 1 μM ionophore A23187 for 10 minutes followed by ionophore removal with cell-free buffer to convert spread platelets to SCIPs (SCIP). Isolated neutrophils (106/mL) were then perfused over the monolayers at 150 s−1 for 5 minutes. (A) Scanning electron microscopy images of spread platelet and SCIP monolayers prior to neutrophil perfusion. (B) Scanning electron microscopy images of neutrophils that have interacted with Spread platelet or SCIP monolayers. (C) Neutrophil-platelet interactions were visualized by phase-contrast microscopy and the proportion of adherent neutrophils exhibiting stationary (i) or spread (ii) morphology quantitated. Images in panels A and B are representative of more than 10 individual experiments. Bar is 5 μm. Results in C are expressed as mean (±SEM; n = 4). *P < .05, **P < .01, ***P < .001.

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