Flow cytometric detection of heterotypic aggregation kinetics. neutrophils (3 × 106 cells/mL) were labeled green (CD45-FITC) and E3-ICAM cells (6 × 106cells/mL) were stained red (LDS-751) for 15 minutes at room temperature. Excess LDS-751 label was removed by centrifugation, cell populations were equilibrated in 37°C buffer containing 1.5 mmol/L Ca2+ for 2 minutes, stimulated with 1 μmol/L FMLP, and sheared in a cone-plate viscometer at a shear rate of 200 s−1. Samples were withdrawn at indicated time points, fixed with 0.5% cold paraformaldehyde, and analyzed on a flow cytometer. (A) Neutrophil and E3-ICAM gated on their characteristic forward versus side scatter. (B) Initial particle distribution at time zero. (C) Heterotypic aggregate distribution 90 seconds after stimulation and application of shear. (D) Kinetics of neutrophil–E3-ICAM aggregation for the experiment depicted in A through C. Dotted and solid lines denote the percentage of neutrophils in heterotypic and homotypic aggregates, respectively. (E) Kinetics of aggregation for a representative experiment where 3 × 106neutrophils/mL were stimulated and sheared with 1.5 × 106E3-ICAM cells/mL. (F) Adhesion efficiency (±SEM) for neutrophil-neutrophil and neutrophil–E3-ICAM collisions calculated from three independent experiments described in D and E.
