Cd61 fitc

The antibodies against CD14-PerCP, CD16-PECy7, CD86- APC, CD61–FITC, CD62P-PE, unlabeled CD62P and FACS™-Lysing solution were obtained from BD Bioscience (San Jose, CA, USA). Thrombin receptor activator peptide (TRAP – 10 µM) was purchased from Sigma Aldrich (St Louis, MO) and abciximab [an antibody against glycoprotein IIb/IIIa inhibiting platelet aggregation (ReoPro®; 2 mg/ml)] from Lilly Benelux S.A/N.V.

Platelets or bone marrow single cell suspensions were stained for flow cytometry analysis as previously described [30 (link), 31 (link)]. Antibodies used were: CD61-FITC, CD41-PE, Sca1-PECy7, CD34-FITC, Lin-cocktail-APC, CD16/CD32-PE, cKit-PerCP, CD71-FITC, Ter119-V405 (BD Pharmingen, Oxford, United Kingdom), Clec2–FITC (AbD Serotec, Kidlington, United Kingdom), CD42a-FITC, CD42b-DL649, CD42c-FITC (Emfret, Wurzburg, Germany), GPVI-PE (R&D, Abingdon, United Kingdom), CD9-PE, CD31-PECy7 (Abcam, Cambridge United Kingdom) and CD49b-PB (BioLegend, San Diego, CA).

Paraformaldehyde was obtained from Affymetrix (Santa Clara, CA). Anti-GITR, anti-GITRL antibodies and the respective isotype control were from R&D Systems (Minneapolis, MN). CD19-FITC, CD41a-PECy5, CD41a-PE, PAC-1-FITC, CD61-FITC and CD62P-FITC were from BD Pharmingen (San Diego, CA), CD3-APC/Fire and CD56-PeCy7 were from Biolegend (San Diego, CA). The goat anti-mouse PE conjugate was from Dako (Glostrup, Denmark). Biocoll Separating Solution was purchased from Biochrom AG (Berlin, Germany). VPA was from Sigma-Aldrich (St. Louis, MO). Thrombin Receptor Activator Peptide 6 (TRAP-6), collagen and ADP were purchased from Sigma-Aldrich (St. Louis, MO).

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.

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).

The CD61 antigen (beta III integrin) is present in all normal, resting and activated platelets. The population of platelets was identified on the basis of particle size (forward and 90° side scatter) and the association with CD61. The obtained gated platelets expressed CD61 on the surface in almost 100% of cases (Figure 1).
Platelets in a measured amount of 2 × 10⁶ cells per 100 µL were incubated with monoclonal antibodies for 30 min at 4 °C. Monoclonal antibodies CD61/FITC (BD, Biosciences, USA), were used for characterization. After incubation, 400 µL of citrate buffer was added to the cells and a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) was used for analysis. Thirty thousand events were accumulated for each sample. Data were collected using the CELLQuest program (Becton Dickinson, Franklin Lakes, NJ, USA).

Colonies expanded from blood were trypsinised, washed and stained with antibodies: CD31-FITC, CD90-PE, CD105-PE (AbD Serotec, Kidlington, UK), CD19-PE, CD33-FITC, CD34-PerCp, CD45-PE-Cy7, CD61-FITC, CD73-PE, (BD Biosciences, Oxford, UK) and CD271-APC (Miltenyi Biotec), at manufacturers recommended concentrations. Cells were washed, 30,000 events captured on a LSRII flow cytometer and the data analysed using FACSDiva Software (both BD Biosciences).