Flow cytometric detection of neutrophil–ICAM-1 cell aggregation.

Neutrophils (1 × 10⁶ cells/mL) were fluorescently labeled green (CD45-FITC) and ICAM-1 300.19 cells (2 × 10⁶ cells/mL) were labeled red (LDS-751) for 10 minutes at room temperature. The 2 cell populations were combined in the cone-plate viscometer and equilibrated to 37°C in buffer containing 1.5 mmol/L Ca⁺⁺ for 2 minutes before stimulation with 1 μmol/L fMLP and initiation of fluid shear. Samples were taken at prescribed time points and immediately fixed in 0.5% cold formaldehyde. Two-color flow cytometry was used to detect distinct populations of single neutrophils (N) and 300.19 ICAM-1 cells (I); homotypic neutrophil doublets (N₂) and triplets (N₃); and 2-color heterotypic aggregates containing a single 300.19 cell bound to 1 (IN), 2 (IN₂), or 3 or more neutrophils (IN₃+). Representative dot plot depicts aggregation of neutrophils with I_high at 600 s⁻¹ at 1 minute after stimulation and initiation of shear. Each dot represents a single particle event containing 1 or more cells.