Fig. 3. PECAM-1 ligation enhances cell-surface expression of 6 integrins on mouse blood neutrophils in vitro as quantified by flow cytometry and observed by confocal microscopy. Blood-derived neutrophils incubated for 30 minutes in wells of 96-well plates, coated with BSA (control) or different combinations of PECAM-1, ICAM-1 and IL-8, were assayed for 6 integrins expression by flow cytometry. The binding of the primary mAb GoH3, relative to the binding of an isotype-matched control mAb, is expressed in terms of relative fluorescence intensity (RFI). (A) Cell-surface expression of 6 integrins on WT blood neutrophils after adhesion to different proteins and protein combinations. (C) Cell-surface expressions of 6 integrins on WT neutrophils incubated in wells in the presence or absence of anti-ICAM-1 or anti-ß2 mAbs, and expression on the cell surface of PECAM-1-deficient neutrophils. Results represent means±s.e.m. from three to six samples in each condition using different neutrophil preparations. Asterisks and crosses indicate significant differences from samples incubated in BSA-coated wells: *P<0.05; **P<0.01; ***P<0.001 (A,C), and significant differences from samples incubated in wells coated with PECAM-1, ICAM-1 and IL-8 (total protein being 10 µg ml^–1): ⁺P<0.05; ⁺⁺⁺P<0.001 (C). (B,D) Representative histograms from experiments shown in A,C, with the hatched histograms representing binding of control IgG. Selected samples were also analysed using a confocal laser-scanning microscope (Zeiss LSM 5 Pascal incorporating a 100x oil A-plan objective with a numerical aperture of 1.25) equipped with an argon laser (excitation wavelength of 488 nm). Expression of 6 integrins was indirectly observed using the mAb GoH3 against 6 integrins and an Alexa-Fluor-488-conjugated secondary antibody. Representative images of mid-level sections of cells from different conditions are shown. (E) WT neutrophils incubated in BSA-coated wells. (F) WT neutrophils incubated in PECAM-1/ICAM-1/IL-8 (10 µg ml^–1)-coated wells. (G) PECAM KO neutrophils incubated in PECAM-1/ICAM-1/IL-8 (10 µg ml^–1)-coated wells. The binding of the anti-6-integrin mAb under different experimental conditions, corrected for the binding of an isotype-matched control mAb and expressed in terms of mean fluorescence intensity (arbitrary units), is also shown (mean±s.e.m. from three to six samples in each condition using at least three different neutrophil preparations). Bar, 10 µm.