Mouse rat ccl5 rantes quantikine elisa kit

Cells were cultured in 12-well plates and stimulated with varying concentrations of OSM (0, 1, 5, 10, 30 and 50 ng/ml), IL-1 (10 ng/ml), IL-6 (30 ng/ml), leukemia inhibitory factor (LIF; 20 ng/ml) or IL-11 (30 ng/ml) for 24 h. All cytokines were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Cells were centrifuged at 6,000 × g and 4°C for 10 min, and supernatants were collected and stored at −20°C prior to analysis.
MCP-1, MIP1α and RANTES levels were respectively determined using the Mouse CCL2/JE/MCP-1 Quantikine ELISA kit (MJE00; R&D Systems, Inc.), Mouse CCL3/MIP1α Quantikine ELISA kit (MMA00; R&D Systems, Inc.), and Mouse/Rat CCL5/RANTES Quantikine ELISA kit (MMR00; R&D Systems, Inc.), following the manufacturer's protocol. Cell supernatants (50 µl) or specific standard substances (MCP-1, MIP1α and RANTES) from the kits were added to the wells of a 96-well plate and incubated for 2 h at room temperature. Biotinylated conjugates from the kits were added to the wells and incubated for 2 h at room temperature. Following rinsing, the substrate avidin peroxidase complex was added and incubated for another 30 min. Reactions were attenuated using stop solution and optical density values were measured at a wavelength of 450 nm using a microplate reader. The concentration of cytokines in samples was calculated via interpolation of a standard curve.