V22887

Before giant plasma membrane vesicle (GPMV) isolation, live cells were incubated with DiD (1:1,000, Invitrogen, V22887), which is a lipophilic fluorescent dye located in the disordered domain, for 10 min at 37^°C, then washed three times with “GPMV buffer” (10 mM HEPES, 150 mM NaCl, 2 mM CaCl₂, pH 7.4), and then incubated at 37°C for about 60 min in GPMV buffer containing 25 mM PFA and 2 mM DTT to induce vesiculation (Levental et al., 2010 (link)). The supernatant was collected and incubated with 0, 1, 10, 30, 100, 200, 500 μM, and 1, 5, 10, 30 mM MβCD or 0, 1, 5, 10, 30 μM 3oc, and then used for three-dimensional (3D) Confocal imaging to analyze the distribution of ordered and disordered domains.

HUVECs were transfected with 100 pmol/L of control RNA or siRNA targeting human PAR-1. Three days after transfection, cells were treated with 10 ng/ml tumor necrosis factor-α (TNF-α) and then exposed to laminar flow (12 dynes/cm²) for 24 hours. Separately, U937 cells were treated with 5 μM Vybrant DiD and incubated for 20 minutes at 37 °C (V-22887, Invitrogen, Carlsbad, CA, USA), as described previously^{59 (link)}. These cells were then washed twice, resuspended in serum-free medium (RPMI), added to HUVECs that had been cultured in 100 mm dishes and incubated for 1 hour at 37 °C (1 × 10⁷/100 mm dish). After removing media, non-adherent cells were removed by washing twice with phosphate buffered saline (PBS). Adherent cells were observed under a confocal fluorescence microscope (Leica, Bannockburn, IL, USA).