Elisa maxtm deluxe set human mcp 1 ccl2

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.

In order to evaluate the anti-inflammatory activity of the CU- and RV-based SLNs, containing or not containing LNA, NCTC 2544 cells were seeded at the concentration of 250 × 10³ cells/well in a 12-well multiwell culture plate. After 24 h, cell culture medium was removed and replaced with fresh culture medium containing or not the empty SLNs (containing only CU or RV) and SLNs loaded with LNA (at a final concentration of 5 µg/mL) in the absence and in the presence of TNF-α (20 ng/mL), as a pro-inflammatory stimulus. At the end of the treatment period (24 h), surnatant was collected and stored at −80°C until the evaluation of the cytokine secretion. The presence of the pro-inflammatory cytokines IL-6 and MCP-1 were evaluated by ELISA assay using commercially available kits (ELISA MAX^TM Deluxe Set Human IL-6, cat. 430504; ELISA MAX^TM Deluxe Set Human MCP-1/CCL-2, cat. 428804, Bio-Legend, San Diego, CA, USA) following the instructions of the manufacturer. The minimum level of measurable cytokines was 4 pg/mL and 3.9 pg/mL, for IL-6 and MCP-1, respectively.