Fura 2 acetoxymethyl am

We purchased 20(S)-ginsenoside Rg3 from the Ambo Institute (Daejon, Korea) and thrombin and all materials for platelet aggregation from Chrono-Log Corporation (Havertown, PA, USA). We purchased all materials for buffer solution and Rp-8-Br-cAMPS, Rp-8-Br-cGMP, p-chlorophenylthio (CPT)-cAMP, 8-Br-cGMP from Sigma (St. Louis, MO, USA) and Fura 2-acetoxymethyl (AM) from Invitrogen (Eugene, OR, USA). Thapsigargin and cAMP/cGMP enzyme immunoassay kit were obtained from Cayman Chemical (Ann Arbor, MI, USA). Anti-IP₃-receptor type I, anti-phosphor-IP₃-receptor type I (Ser¹⁷⁵⁶), anti-extracellular signal-regulated kinase (ERK) (1/2), anti-phosphor-ERK (1/2), anti-rabbit IgG-horseradish peroxidase, and cell lysis buffer were obtained from Cell Signaling (Beverly, MA, USA). Anti-β-actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal to CD62P (p-selectin) antibody was purchased from Biolegend (San Diego, CA, USA). Polyvinylidene difluoride (PVDF) membrane and enhanced chemiluminesence solution (ECL) were purchased from General Electric Healthcare (Chalfont St. Giles, Buckinghamshire, UK).

5 μM Fura 2-acetoxymethyl (AM) (calcium indicator, Invitrogen) was loaded into HEK-293 cells stably expressing hDRD1 with the incubation in an imaging buffer (in mM: NaCl 140, KCl 5, MgCl₂ 1, CaCl₂ 2, HEPES 10, Glucose 10, 0.1% Pluronic F-127, pH 7.4) at 37°C for 30 min and then washed with the imaging buffer. The cleavage of the AM ester group was allowed by the incubation for 1 h at 37°C. The fluorescence signal was measured at 510 nm by dual excitation at 340 nm and 380 nm using a spectrofluorophotometer (Tecan) upon the addition of various concentrations of neurotransmitters and 100 μM of ATP. For intracellular Ca²⁺ assay of nanovesicles, hDRD1 expressing nanovesicles were produced from Fura 2-AM loaded HEK-293 cells stably expressing hDRD1 according to the process described above. The Fure 2-AM loaded nanovesicles were immobilized on poly-D-lysine treated 96-wells plate with the incubation at 37°C for 2 h.