Acea novocyte 3000

For externalized phosphatidylserine (PS) analysis, washed platelets were incubated with VSV-G or Spike pseudoparticles (1:10) for 10 min followed by activation with collagen (30 μg/ml) and thrombin (0.5 units, Sigma-Aldrich, Burlington, MA, United States) or vehicle for 15 min (350 rpm, 37°C; BioShake IQ). Platelets were stained with Annexin V-Pacific Blue (1:50; BioLegend, San Diego, CA, United States) and CD61-APC (1:100; BioLegend, San Diego, CA, United States) in annexin binding buffer for 20 min (350 rpm, 37°C; BioShake IQ). For Ca2⁺ levels analysis, washed platelets were stained with Fluo-4 AM (5 μM, Thermo Fisher Scientific, Waltham, MA, United States) for 30 min at 37°C followed by incubation with pseudoparticles (1:10) for 10 min. Platelet samples were activated with vehicle or collagen (30 μg/ml) for 15 min.
In both cases, samples were diluted with modified Tyrode’s HEPES buffer and analyzed on the ACEA Novocyte 3,000 (ACEA Biosciences, San Diego, CA, United States). Analysis was performed using the FlowJo software v.10 (TreeStar, Ashland, OR, United States).

Cell cultures were washed in blocking-PBS buffer [1% Bovine Serum Albumin (Sigma-Aldrich) and 1% FBS in PBS 1X (Thermo Fisher Scientific)], stained with primary-conjugated antibodies (Additional Table 3), as well as with 0.2 μg/mL 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI; Sigma-Aldrich) to select viable cells. Flow cytometry data were analyzed with ACEA Novocyte 3000 (Acea Bioscience Inc., USA), and results were processed with NovoExpress Software (Acea Bioscience Inc.)