Culture supernatants of HUVECs, harvested for quantification of mRNAs, were collected and stored at −80 °C for subsequent quantification of cytokine content by ELISA: specific kits for IL-8 and VEGF-A (Raybiotech) were used following the manufacturers’ instructions. When required, cells were pre-incubated for 30 min with 10 μM KG-501 or 100 nM QNZ before the exposure to TpF1.
Freshly isolated T-cells (8 × 105/ml) were co-cultured with HUVECs or autologous monocytes (4 × 105/ml) in the presence of 10 μg/ml TpF1 or 0.5 μg/ml tetanus toxoid (GSK, London, UK). After 24 h, the content of IL-8, CCL-20, TF, CCL-1, CCL-13, angiopoietin-1 and angiopoietin-2 in the culture supernatants was quantified by specific ELISA kits (IL-8: Affymetrix, eBioscience, Santa Clara, CA, USA; CCL-20, CCL-1, angiopoietin-1, angiopoietin-2: R&D Systems, Minneapolis, MN, USA; TF: Sekisui Diagnostics, Pfungstadt, Germany; CCL-13: Thermofisher Scientific, Waltham, MA, USA).
Freshly isolated T-cells (8 × 105/ml) were co-cultured with HUVECs or autologous monocytes (4 × 105/ml) in the presence of 10 μg/ml TpF1 or 0.5 μg/ml tetanus toxoid (GSK, London, UK). After 24 h, the content of IL-8, CCL-20, TF, CCL-1, CCL-13, angiopoietin-1 and angiopoietin-2 in the culture supernatants was quantified by specific ELISA kits (IL-8: Affymetrix, eBioscience, Santa Clara, CA, USA; CCL-20, CCL-1, angiopoietin-1, angiopoietin-2: R&D Systems, Minneapolis, MN, USA; TF: Sekisui Diagnostics, Pfungstadt, Germany; CCL-13: Thermofisher Scientific, Waltham, MA, USA).