Cd154 pe

Human IgD was purchased from Abcam (Cambridge, MA, USA). Anti-CD3 and CD28 antibodies were provided by T&L Biological Technology (Beijing, China). A770041 was purchased from Axon Medchem (Groningen, Netherlands). rhTNFR:Fc fusion protein was purchased from Guojian Pharmaceutical Company (Shanghai, China). Anti-mouse IgD antibodies were purchased from eBioscience (San Diego, CA, USA). Anti-human CD69-PE-cy5, CD154-PE, CD4-FITC, CD25-APC, IL-4-APC, IFN-γ-PE-cy7, IL-17-PE, and FoxP3-PE, along with anti-mouse CD3e-PerCP-Cy^TM5.5, CD4-FITC, CD25-APC, CD154-PE, IFN-γ-PerCP-Cy^TM5.5, IL4-APC, IL-17-PE, and FoxP3-PE antibodies were provided by BD Pharmingen (San Diego, CA, USA). Anti-Lck, anti-phospho-ZAP70, anti-phospho-Lck, anti-ZAP70, and anti-β-actin were purchased from Affinity Biosciences, Sigma and Abcam.

Cells were briefly washed in cold PBS+5% FBS, and stained for extracellular markers: CD4-APC (Biolegend, Cat# 300514), CD25-PE (302606, Biolegend), CD134-PE (555838, BD Pharmingen), CD154-PE (555700, BD Pharmingen), LFA-1-PE (1433, Immunotech). Cells were analyzed using the BD Accuri C6 flow cytometer and data analyzed using FCS Express vs 3.0.

Washed platelets were pre-incubated with vehicle control, Akt1/2 inhibitor (Akti 1/2, 5μmol/L, sigma), or p38 MAP kinase inhibitor (SB203580, 10μmol/L, Cell Signaling) or left alone for 5 min at 37°C. Afterwards, platelets were stimulated with ADP (20 μmol/L) for an additional 15 min at 37°C. Samples were fixed in 1% buffered paraformaldehyde and then stained with antibodies against P-selectin-FITC (BD Bioscience), CD147-PE (eBioscience), CD154-PE (Pharmingen), or isotype control antibodies and analyzed by flow cytometry (FACSCanto II, BD Biosciences).