Spheroparticles

HTS Transwell 96-well permeable supports with an 8.0-µm pore polyester membrane (Corning) were used for chemotaxis assays of ED40515(+) and KOB. 235 µl of culture medium containing the indicated dose of chemokine was added to the lower chamber, followed by the addition of the Transwell inserts and the addition of cells in growth medium (75 µl) at a concentration of 5 × 10⁵ cells/ml into the upper chamber. ChemoTx (5 µM pore; Neuro Probe) was used for chemotaxis assays of 32Dβ. 300 µl of chemokine-containing medium was added to the lower chamber, and 55 µl of cells in growth media (2 × 10⁶ cells/ml) were added to the upper chamber. After incubation for 2 h at 37°C in 5% CO₂, the upper chamber was removed and 50 µl of 1× PBS with a 1:100 dilution of SPHERO particles (SpheroTECH) and 1:2,500 dilution of 10 mg/ml ethidium bromide was added into each lower chamber. The numbers of cells and SPHERO particles in lower chamber were quantified with a FACSCalibur (BD), and the numbers of cells that had migrated in each condition were normalized based on the SPHERO particle count.