Recombinant human l selectin

Cells were detached with PBS and counted with Cellometer Vision CBA (Nexcelom Bioscience) imaging cytometer. Depending on experiment PBS containing 2% FBS either with or without Ca²⁺ and Mg²⁺ was used as suspension and wash buffer. Within an experiment the number of cells was adjusted to be equal in all samples. CD24 and GD2 APC (BioLegend) and CD44 PE (BD Pharmingen Biosciences) were used according to manufacturer’s instructions and surface staining was measured using a FACS Calibur or LSRFortessa (Becton Dickinson) flow cytometer and analyzed using FlowJo software (TreeStar). Monoclonal mouse α-human PSGL-1 (EMD Millipore, clone KPL1 and R&D Systems, clone CHO131) was detected by APC α-mouse Ig (BD Pharmingen Biosciences). For selectin binding assays human recombinant selectin-Fc chimera or control IgG Fc fragment (Jackson ImmunoResearch) at 10 μg/ml were incubated 30 min at room temperature (RT) with dissociated cells. After washing the cells APC-conjugated α-human IgG Fc (BioLegend) antibodies were added and incubated for 30 min. Bound selectins were measured as above. Recombinant human L-selectin, P-selectin and E-selectin Fc chimeras were purchased from R&D Systems.

For the generation of P-selectin-coupled beads polybead-carboxylate-microspheres (2 µm, 2.7% solids (w/v) Polysciences), polylink protein coupling kit (Polysciences), and recombinant human P-selectin protein (carrier free, Catalog no. ADP3-050, R&D Systems) were used. Coupling was performed according to the manufacturer’s protocol. About 3.1 mg microparticles were centrifuged (1000g, 10 minutes) resuspended in polylink coupling buffer twice before 21-mg carbodiimide was added for activation. After 15 minutes, 50-µg P-selectin was added and incubated for 120 minutes. Then, beads were centrifuged (1000g, 10 minutes) and washed in storage buffer twice and stored at 4 °C in 100-µL storage buffer. As controls uncoupled beads (without protein incubation) and L-selectin-coupled beads were generated using recombinant human L-selectin (R&D Systems).