VEGF Assay

The VEGF assay was adapted from Gaudry et al.²³ Briefly, 96-Well plates were precoated with 50 µL 0.01% collagen I (Sigma Aldrich, Schnelldorf, Germany) for at least 6 h at RT. Collagen I was removed and the coated surface was dried overnight. The wells were rinsed with sterile tissue culture grade water before transferring cell suspension. 1×10⁶ Neutrophils in 100 µL RPMI medium supplemented with 10% FCS were seeded in a 96 well plate. SBDS (0, 10, 50 µg/mL) were added and incubated for 30 min at 37 °C in an atmosphere containing 5% CO₂. To investigate whether SBDS can prevent VEGF release, neutrophils were stimulated with 50 ng/mL PMA for 2 h. To investigate whether SBDS induces VEGF release, cells were left untreated for 2 h. Cells were centrifuged (300 g, 5 min, RT), supernatant was collected and the concentration of VEGF determined by ELISA (Thermo Fisher Scientific, Germany), as recommended by the supplier. For analysis, the absorbance values from standard and from test samples were corrected with the blank (standard: assay buffer; samples: medium with SBDS), since the values of the controls decreased with increasing concentration of SBDS. The concentration of VEGF was extrapolated using the standard curve.