Sb225002

Pure neutrophils were diluted to the density of 6 × 10⁵ cells/mL in HBSS medium and seeded in 96-well plate with 100 μL in each well. Neutrophils were incubated with different concentrations of drugs (CXCR2 antagonist SB225002, EMD Millipore, Billerica, MA; PI3K inhibitor LY294002 or theophylline, Sigma-Aldrich, St. Louis, MO) for specific time periods (30 min, 90 min, or 150 min) in the incubator at 37°C under 5% CO₂. After incubation, the well plate was centrifuged to remove medium, and the cells were incubated with 100 μL of 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, MO) solution for 2 h. The water-insoluble purple formazan crystals only produced by the living cells were dissolved in 150 μL of dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO). The plate was placed on the orbital shaker for 20 min to facilitate complete crystal dissolution. Finally, 100 μL of DMSO solution was transferred to another new 96-well plate for UV-Vis absorption measurements. Optical density was monitored at 570 nm, with 655 nm as a reference, using a microplate reader (Bio Tek, Winnoski, VT), and the cell viability was calculated using equation (1). The data in each condition were recorded from five different blood samples (donors).

CXCR2 antagonist SB225002 (Sigma) was administered at 7.5 mg/kg intraperitoneally 30 mins before U-grafts implantation, and then every day until day 7. Neutrophil recruitment at day 2 and graft vascularization at day 7 were measured as described before. Daily injection of saline served as control. In selected experiments, SB225002 treatment was combined with rat anti-mouse Ly-6G treatment (clone 1A8; administered as described above) to study the combined effect on U-Graft vascularization.