PLPs coincubation with HUVECs and THP-1 cells. (A) THP-1 cells, treated with 1 μg/mL Pam3CSK4, were coincubated with PLPs for 24 hours and stained with CD41a-FITC, CD42b-FITC (open curve; solid line) for flow cytometry analysis. Isotype IgG (gray filled curve), control (no PLPs; open curve, dotted line). (B) The same experiment was carried out staining THP-1 cells for CD11b-APC, CD41a-FITC, and CD42b-FITC. Samples were then mounted on coverslips for confocal microscopy (PLP). IgG control (isotype) and THP-1 alone (control). Scale bar denotes 10 μm; 100× objective lens. (C) Confocal microscopy also was performed on PKH67-labeled PLPs (green fluorescence) cocultured for 24 hours with THP-1 cells to demonstrate PLP internalization. Blue nuclear staining was performed with 4,6-diamidino-2-phenylindole (DAPI). Scale bar denotes 10 μm; 60× objective. (D) The experiment was performed as described in panel A using CD11b-FITC. Untreated cells (control), activated cells (PAM), PLP (THP-1 cells cocultured with PLPs). (E) HUVECs were treated for 10 minutes with 0.5 U/mL thrombin and then coincubated with PKH67-labeled PLPs for 30 minutes (gray filled curve) and analyzed by flow cytometry. Control (open curve, dotted line), untreated HUVEC + PLPs (open curve, solid line). (F) Confocal microscopy of the experiment described in panel E with additional DAPI staining. HUVECs incubated with PKH67-labeled PLPs (ii-iv) and unlabeled PLPs (i). Scale bar denotes 20 μm; 40× objective.