Fitc conjugated anti cd62p p selectin antibody

Sanguinarine (13-methyl(1,3) benzodioxolo (5,6-c) -1,3-dioxolo(4,5-i) phenanthridinium), (HPLC ≥ 98%, SA, Figure 1) (Absin, Shanghai, China), was dissolved in 0.1% DMSO (final concentration). The EDTA, prostaglandin E1(PGE1) and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO, USA). The CHRONO-LUME reagent and collagen were acquired from Chrono-Log Corp. (Havertown, PA, USA). The collagen-related peptide was obtained from Dr. Newman’s lab (Blood Center of Wisconsin, Milwaukee, WI, USA). The FITC-conjugated anti-CD62P (P-selectin) antibody and FITC-conjugated anti-PAC-1(αIIbβ3) antibody were purchased from Biolegend (San Diego, CA, USA). The Fluo-3AM was purchased from MCE (Shanghai, China). The CellTrace Calcein Green was purchased from Invitrogen (Carlsbad, CA, USA). The anti-phospho-PI3K(Ser1070), anti-PI3K, anti-phospho-Akt(Ser473), anti-phospho-GSK3β (Ser9), anti-phoshpo-PLCγ2 (Tyr1217), anti-β3, anti-phospho-β3 (Tyr474), anti-Src, anti-phospho-Src (Tyr416) and anti-phospho-Syk (Tyr525/526) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-Syk, anti-PLCγ2, anti-Akt and antil-GSK3β were acquired from Santa Cruz Biotechnology (Dallas, TX, USA).

The release of P-selectin from the α-granules was quantified by flow cytometry as described earlier^{19 (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 FlowJo^{22 (link)}.
Secretion of ATP from the dense granules was assessed by a luminescence method using a luciferin/luciferase kit from Chrono-Log Corporation^{19 (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.