Platelet Isolation and Counting Protocol

Mice were anesthetized with isoflurane and killed by cervical dislocation. Subsequently, the thoracic cavity was opened and the right ventricle was punctured with a syringe, which was prefilled with 200 μL of sterile sodium citrate buffer. Blood was carefully aspirated and transferred into 2 mL tubes, prefilled with 250 μL acid-citrate-dextrose (ACD) buffer and 250 μL modified tyrode’s buffer (MTB). Samples were spun down in a swing-out centrifuge for 6 min at 200 G at room temperature (RT) with breaks turned off. The upper platelet rich plasma (PRP) was transferred while the remaining sample was washed again with 500 μL MTB, repeating the procedure. Collected PRP was spun down for 6 min at 600 G, supernatant discarded and platelets resuspended in media. Platelets were manually counted at least twice to adjust concentration between conditions (WT vs Cxcl4^−/−) under a microscope using a Neubauer chamber and Trypan-Blue staining. Further incubation was carried out in DMEM (+10% FCS, +1% Penicillin/Streptomycin) in an CO₂ incubator at 37°C with 5% CO₂ gas supplement and >90% humidity.