Cd62p fitc

Thrombin was purchased for Sigma. Human IFN-β was purchased from PBL Interferon Source. Mouse IFN-β was purchased from R&D Inc. Anti-human CD42a-PeCP, CD62P-FITC, CD63-PE, and FITC conjugated PAC-1 antibodies (recognize active form of human αIIbβ3 integrin) were purchased form BD Biosciences. Anti-mouse CD31-PE, CD41-PE, CD61-FITC, CD62P-Biotin, CD86-PE antibodies, and Streptavidin-APC were purchased form BD Biosciences. Anti-mouse F4/80-APC and CD41-APC antibodies were purchased from eBiosciences. Anti-mouse CD11b-AF488, and MHC class II-PE-Cy5 antibodies were purchased from Biolegend. PE conjugated JON/A antibodies (recognize active form of mouse αIIbβ3 integrin) and fluorescently labeled anti-CD42c antibodies for in vivo labeling of mouse platelets were purchased from Emfret Analytics Inc. Fura-2M probe, a membrane-permeable derivative of Fura-2, was purchased from Molecular Probes. GA (Copaxone™) was purchased from Teva. Undiluted Copaxone (20 mg/ml) was stored at +4°C. For experiments GA was diluted to proper concentrations in PBS, stored at room temperature and used within 12 hours.

Immediately after isolation, 2 µl mouse platelets were incubated with antibodies against IgG1-FITC (Milteny Biotec, Bergisch Gladbach, Germany, order no.: 130-098-847, 1:25) or CD62P-FITC (BD Biosciences, Heidelberg, Germany, Cat: 561923, 1:25). Human platelets were incubated with antibodies against FITC Mouse IgG1 (BD Biosciences, Cat: 555748, 1:25) or FITC Mouse Anti-Human CD62P (BD Biosciences, Cat: 555523, 1:25). Subsequently, 46 µl of FACs buffer (2 mM EDTA, 0.5 % FCS ad 100 ml PBS, pH 7.1) was added and the samples incubated for 30 min at 4 °C in dark. To study apoptosis, 2 µl of platelets were incubated with 5 µl Annexin V-FITC (Milteny Biotec, Order no.: 130-097-928) in 100 µl Annexin V Binding Buffer (Miltenyi Biotec, 20x Stock Solution) at RT for 30 min in dark. Immediately before analysis, 2 µl of 7-AAD (Miltenyi Biotec) was added. All samples were centrifuged at 300g for 10 min and the pellets were resuspended in 100 µl of FACs flow (BD FACSFlow). FACs analysis was instantly performed with a BD FACScan.

Paraformaldehyde was purchased from Affymetrix (Santa Clara, CA). Anti-human TACI antibody (clone 165604) and the respective isotype control were from R&D Systems (Minneapolis, MN). CD41a-PeCy5, CD61-FITC and CD62P-FITC were from BD Pharmingen (San Diego, CA). The goat anti-mouse PE conjugate was from Dako (Glostrup, Denmark). Bicoll Separating Solution was purchased from Biochrom AG (Berlin, Germany). Recombinant human BAFF (rhBAFF) and recombinant human APRIL (rhAPRIL) was from PeproTech (Rocky Hill, NJ, USA). Thrombin Receptor Activator Peptide 6 (TRAP-6), collagen and ADP was purchased from SigmaAldrich (St. Louis, MO). Citrate buffer contained 10 mM sodium citrate, 150 mM NaCl, 1 mM EDTA, 1% dextrose, pH 7.4. GI254023 was from Tocris (Bristol, UK) and Batimastat was from Calbiochem (Darmstadt, Germany).

All chemicals were purchased from Sigma Chemical (St Louis, MO, USA). The HEPES buffer solution consisted of 137 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl₂, 5.6 mM glucose, 20 mM HEPES, 1 mg/mL bovine serum albumin, 3.3 mM NaH₂PO₄, pH 7.4. The lysing solution contained 155 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA, pH 7.4 and was stored at 4 °C. The antibodies against surface markers were: CD4-PE, CD14-PE, CD45-APC, CD61-PerCP, CD62P-PE, CD62P-FITC, CD63-PE, CD66b-FITC, PAC-1 FITC, and their isotype control antibodies (all from BD Pharmingen, San Jose, CA, USA). CD61-APC was from Dako (Glostrup, Denmark).

Mouse platelets were isolated as reported previously12 (link). Briefly, mice were anaesthetized by an intraperitoneal injection of Ketamine 100 mg/kg and Xylazine 10 mg/kg (AniMedica). Blood drawn directly from the heart was immediately collected in ethylenediaminetetraacetate (EDTA) tubes (S-monovettes, Sarstedt, Germany). Immediately after isolation, 5 μl EDTA blood was mixed with 95 μl FACS buffer (2 mM EDTA, 0.5% FCS ad 100 ml PBS, pH 7.1) and 16 μg/ml Thiazine Red and incubated for 3 hours at 4 °C. Then the cells were lysed using Miltenyi red blood cell lysis buffer, centrifuged and the pellet dissolved in 100 μl FACS flow. Some samples were incubated just prior the lysis with 5 μl of IgG1-FITC control (Miltenyi 130-089-867), CD62P-FITC (BD Biosciences, Heidelberg, Germany, Cat: 561923), CD31-FITC (Miltenyi 130-097-424) or CD61-FITC (Miltenyi 130-098-722) for 15 min at room temperature. FACs analysis was instantly performed with a BD FACScan. For the aggregation assays, isolated platelets were incubated with or without 2 mM CaCl₂ for 20 min at 37 °C, then fixed with 4% PAF, and incubated with 16 μg/ml Thiazine Red for 2 hours at 4 °C, cells were centrifuged and visualized under the microscope or in the FACS.

HYW is the experience prescription of professor Yu Rencun, a national famous old Chinese medicine doctor. It is also the in-hospital preparation of Beijing Hospital of Traditional Chinese Medicine affiliated to Capital Medical University (Beijing Pharmaceuticals, Z20053296). It is composed of 15 kinds of Chinese herbal medicines, including Paeoniae Radix Rubra, Curcumae Radix, Ginseng Radix et Rhizoma Rubra, and Astragali Radix [14 (link)]. The medicinal materials were provided by the pharmacy of Beijing Chinese Medicine Hospital. Aspirin was purchased from Sigma, USA; TEG, thromboelastography system, kaolin solution, and calcium chloride solution were purchased from Haemonetics, USA; CD62P-FITC, IgG1-FITC, and CD41-PE were purchased from BD Biosciences; and VEGF, CD62P, bFGF, TGF-β, and PDGF-AA ELISA kit were purchased from US/Canada RD Corporation.