The release of P-selectin from the α-granules was quantified by flow cytometry as described earlier19 (link). Briefly, the platelets were isolated from PRP by centrifugation, washed twice and finally resuspended in HEPES-buffered Tyrode’s solution without calcium, pH 7.4 containing 0.2% bovine serum albumin. Washed platelets (1–1.5 × 106) were incubated with 2 micro liters of FITC conjugated anti-CD62P (P-selectin) antibody (BioLegend, #304904) solution for 15 min at 37 °C without stirring. Expression of P-selectin on platelet surface was quantified by flow cytometry (BD LSR II, BD Biosciences) and analyzed by the FACSDiva or FlowJo22 (link).
Secretion of ATP from the dense granules was assessed by a luminescence method using a luciferin/luciferase kit from Chrono-Log Corporation19 (link). The luciferin/luciferase reagent was added to platelets one minute prior to addition of collagen. Platelet aggregation was monitored by a standard optical density method using a Lumi-Aggregometer from Chrono-Log Corporation.
Secretion of ATP from the dense granules was assessed by a luminescence method using a luciferin/luciferase kit from Chrono-Log Corporation19 (link). The luciferin/luciferase reagent was added to platelets one minute prior to addition of collagen. Platelet aggregation was monitored by a standard optical density method using a Lumi-Aggregometer from Chrono-Log Corporation.
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