Cd41a pe

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).

On day 19 of cultures, the methylcellulose cultures were washed with PBS. Living cells were counted in trypan blue and stained with the following antibodies in PBS 4%BSA at 4 °C 45 min: CD45Pe-Vio770 (Miltenyi), CD34Vioblue (Miltenyi), CD41aPE (BD), CD43FITC (Miltenyi), CD31FITC (Beckman Coulter), CD235aAPC (BD), IL1-RAP APC (R&D), CD38PE (Miltenyi), CD71PE (BD), CD14FITC (Beckman Coulter), CD33APC (BD), CD133APC (BD), BB9PE (BD), SSEA1PE (BD). After 1 h, cells were washed and resuspended in PBS 4%BSA with viability staining reagent 1 µg/mL (7-AAD) 7-aminoactinomycin D (Sigma Aldrich). Stained cells were analyzed with a MACSQuant 10 (Miltenyi Biotec) flow cytometer and Flowjo analysis software.