Human il 6 elisa kit 2

Conditioned media were prepared by incubating cells for 48 h as described above. The CM were analyzed using the Human IL-6 ELISA Kit II and reagents, following the procedures described by the manufacturer (BD OptEIATM; 550799).

The day before irradiation, cells were placed in 24-well plates at density 4 × 10⁵ cancer cells per well (monocultures) or 2 × 10⁵ cancer cells and 2 × 10⁵ normal cells per well (co-cultures). Immediately after, irradiation medium was changed and left for 72 h. The type and quantity of cytokines and chemokines secreted by cells into conditioned medium were assessed using LEGENDplex™ Human Inflammation Panel 1 (analyzed cytokines and chemokines: IL-1β, IFN-α2, IFN-γ, TNF-α, MCP-1, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-18, IL-23, IL-33; BioLegend) according to the manufacturer’s instruction. Samples were analyzed on BD FACSCanto II flow cytometer. Raw data were analyzed using LEGENDplex^TM Data Analysis Software Version 8.0 (BioLegend, San Diego, CA, USA). Furthermore, IL-6 and MCP-1 were examined using Human IL-6 ELISA Kit II (BD Biosciences, Franklin Lakes, NJ, USA) and ELISA MAX^TM Deluxe Set Human MCP-1/CCL2 (BioLegend, San Diego, CA, USA) according to the manufacturer’s instruction. Absorbance was measured on Microplate Reader.