Quantification of monocytes, PMNs, and T and B lymphocytes adhering to platelet thrombi and platelet monolayers. The experimental conditions for the whole blood perfusions are described in the legend to Fig 1. The leukocytes adhering to platelets were detached with EDTA and fixed in 0.5% paraformaldehyde-PBS solution. The leukocyte suspension was then diluted in modified Tyrode buffer before incubation with leukocyte type-specific antibodies (see Materials and Methods). In addition, a fresh blood sample was taken at the start of the perfusion experiments and treated with FACS lysing solution before the addition of antibodies. Data (1,000 events) were acquired on an FSC versus SSC dot plot, in which the leukocyte population was gated. Using a panel of PE- and FITC-labeled leukocyte type-specific antibodies (see Materials and Methods), the leukocyte types were quantified in a FL1 (FITC signal) versus FL2 (PE signal) dot plot mode. Shown is the distribution of leukocyte types adhering to (A) platelet thrombi () and (B) platelet monolayers () in comparison with the leukocyte distribution in fresh blood samples (□). The data represent the average values ± SD of 7 donors (monolayers) and 13 donors (thrombi). Statistical analysis by paired Student's t-test: *, P < .01; **, P < .0001.