Evaluating Platelet Activation and Reactivity

The release of platelets’ calcium ions provides a sensitive tool to evaluate platelets’ activation and reactivity [72 (link)]. Isolated platelets (read above for platelets isolation) were resuspended in 1 mL of Tyrode’s buffer at the concentration of 1 × 10⁶ platelets/mL. Platelets were seeded in a glass bottom dish (Ibidi, Gräfelfing, Germany) and incubated with 10 µM Fluo-4 AM (Thermo Fisher, Monza, Italy), 0.1% (w/v) Pluronic F-127 (Sigma Aldrich, Milan, Italy). Platelets without and with the addition of 400 ng/mL of PFOA were activated with 10 µM TRAP-6. Platelets were analyzed in time-lapse, with DMI6000CS fluorescence microscope and 40×/0.60 dry objective magnification (Leica Microsystem, Wetzlar, Germany) for 30 min at 37 °C. Images were analyzed in real time using a differential interference contrast (DIC) and fluorescence objectives. Samples were acquired using a DFC365FX camera and processed using the Leica Application Suite (LAS-AF, Wetzlar, Germany) 3.1.1. software (Leica Microsystem, Wetzlar, Germany).