P or e selectin human igg fc chimeric proteins

Staining for surface antigens was performed in PBS/1% fetal bovine serum for 15 minutes at 4°C. For tetramer binding, cells were incubated for 45 minutes at room temp. P/E-selectin binding was determined by incubating cells with P- or E-selectin human IgG Fc chimeric proteins (R&D Systems) for 30 minutes in 1% FBS/Dulbecco’s PBS containing Ca²⁺ and Mg²⁺ (GIBCO) at room temperature. Selectin binding was detected using anti-human IgG Fc phycoerythrin (eBioscience). Cells were then stained with fluorescent antibodies as described above. Data was acquired using either a BD LSRII Flow Cytometer in the OHSU Flow Cytometry Core Facility. Flow cytometry data was analyzed using FlowJo software, version 9.9 or 10.