Dxp11

The chemoattractant activity of SDF was measured by a cell migration assay using Jurkat cells, an immortalized T cell line. SDF or SDFNP were diluted in 0.6 mL migration medium (RPMI-1640 medium containing 0.5% BSA) and added to wells in a 24-well plate (bottom wells). After 30 min incubation at 37°C, transwell inserts (polycarbonate, pore size 5 μm, diameter 6.5 mm, Corning) were placed into the wells and loaded with 5×10⁵ Jurkat cells in 100 μL migration medium. The plate was incubated at 37°C for 2 h followed by removal of the transwells. Migrated cells in the bottom wells were mixed with fluorescent counting beads (50 μl/well, Invitrogen^™ 123count eBeads) and counted by flow cytometry (DXP11, BD Biosciences) at high speed for 60 sec. For negative controls, the cells were added to the transwells with migration medium in the bottom wells. For the input cell number, cells were added directly to the bottom well and counted. The ratio of the cell-to-bead number was used for calculation, and migrated cells were presented as the percentage of the input after subtraction of that in negative controls.