2.4. ELISA CXCL8/IL-8

The determination of the cytokine CXCL8/IL-8 was carried out using the commercial kit Quantikine® ELISA from R & D Systems. Both the controls, samples, and standards were quantified in duplicate. Reagents and standards were prepared as described by the manufacturer. 100 μL of the RD1-85 diluent was added to each well of the 96-well format plate. Then, 50 μL of standard and samples (supernatants of diluted cell cultures 1/100) per well were added and subsequently incubated for 2 hours at room temperature. After the incubation period, the total reaction was removed by aspiration and washed 4 times with Wash Buffer (400 μL). 100 μL of conjugate CXCL8/IL-8 reagent was added to all wells and incubated for 30 minutes at room temperature. It was again washed 4 times with Wash Buffer. At the end of this stage, 200 μL of Substrate Solution is added to each well and left at room temperature for 30 minutes protected from light. After this incubation period, 50 μL of Stop Solution per well was added and the optical density was determined at 450 nm.