Endothelial-Platelet Interaction Assay

Endothelial cells were plated at a density of 2.5 × 10⁴ cells in a 96-well plate, and after 24 h were pretreated 1 h before and during the experiment with the non-specific pharmacological TRPM7 blockers, 2-Aminoethoxydiphenyl borate (2-APB) (100 µM (Sigma, USA)) and Carvacrol (0.732 M, (Sigma, USA)), the TRPM7 α-kinase domain inhibitor TG100-115 (1 µM (STCB, USA)), the vWF inhibitor Caplacizumab (0.8 µg/mL, (MCE, USA)), the ICAM-1 inhibitor A-286982 and A-205804 (25 nM and 25 nM, respectively (STCB, USA)), or the P-Sel inhibitor KF38789 (50 nM (STCB, USA)), or transfected with an siRNA against TRPM7 (siRNA^TRPM7) or a non-targeting siRNA (siRNA^Nontarget). In brief, cells were plated overnight in 96-well or 6-well plates and transfected with 5 nmol/L siRNA (Dharmacon) using lipofectamine (Invitrogen) and Opti-MEM (Gibco) according to manufacturer instructions for 4 h. Experiments were performed 48 h after transfection. To start the assay, LPS (O127:B8, Sigma-Aldrich) was added at a final concentration of 20 µg/mL with fluorescent-labeled platelets (~ 2.25 × 10⁶ platelets per well, contained in 100 µL of minimal experimentation medium) for 24 h at 37 ℃. Platelets were stained with the green fluorescent dye vibrant DiO (ThermoFisher Scientific) for 15 min at 37 °C. Then, non-adherent platelets were washed thrice with warm phosphate-buffered saline (PBS) and observed in the FLoid Cell Imaging Station (ThermoFisher Scientific). This experiment was performed in technical triplicate, and six fields for each condition were analyzed.