Recombinant murine rantes ccl5

BMMC-conditioned cell-free supernatants were prepared using sensitized (0.5 μg mouse anti-DNP IgE/ml/2×10⁶ BMMC, 12h, 37°C) BMMC treated with JTE-013 (1 μM) or vehicle for 30 min at 37°C prior to adding Ag (DNP-HSA, 100 ng/ml/30×10⁶ BMMC). After 24h, supernatants were collected and stored at −80°C. Splenic CD4⁺ and CD8⁺ T cells were isolated using the EasySep kit (StemCell Technologies, Vancouver, BC, Canada) and used together for migration experiments. T cell enrichment was verified by flow cytometry. Assays were conducted using 5 μm pore size polycarbonate filters in Costar 24-well Transwell chambers (Corning, Corning, NY) with 850 μl BMMC-conditioned media in the lower chamber and 10⁵ T cells in 200 μl in the upper chamber. Recombinant murine RANTES/CCL5 (100 ng/ml, Peprotech, Rocky Hill, NJ) and medium, DMSO vehicle or JTE-013 were used as positive and negative controls, respectfully. After 24h at 37°C, T cells in the lower chambers were counted in quadruplicate using flow cytometry after propidium iodide staining to exclude dead cells from analysis (FACScalibur, BD Biosciences). Migration was expressed as fold of control (culture medium alone).