Streptavidin conjugated horseradish peroxidase

The release of human CX3CL1 and CXCL16 into the supernatant was analyzed by ELISA. Before the measurement, the culture supernatants were concentrated from 3 ml to 0.5 ml using Vivaspin 6 columns (10.000 MWCO) (Sartorius, Göttingen, Germany). 96 well plates were coated in PBS overnight at 4°C with a capture antibody against CX3CL1 (4 μg/ml) or CXCL16 (1 μg/ml), respectively. Before adding the samples, unspecific binding to the plate was blocked with washing buffer (PBS + 0.05% Tween) containing 2% BSA for 2 h at room temperature. Samples were incubated for another 2 h at room temperature, and the bound chemokines were detected with a biotinylated secondary antibody against CX3CL1 (0.3 μg/ml) or CXCL16 (0.5 μg/ml). The chromogenic reaction was mediated by a standard procedure using 0.1 U/ml streptavidin-conjugated horseradish peroxidase (Roche, Basel, Switzerland) and the BM Blue POD substrate (Roche).

Unstimulated or cytokine-stimulated cells were incubated for 24 h. Conditioned media were harvested and cleared by centrifugation (10 min, 4 °C; 16 000 g). Released soluble, JAM-A (JAM-A ELISA kit, SinoBiological, Beijing, China) or IL-8 (Human IL-8/CXCL8 DuoSet ELISA, R & D Systems, Minneapolis, USA) were quantified per ELISA essentially as recommended by the manufacturers. The chromogenic reaction was mediated by a standard procedure using 0.1 U/ml streptavidin-conjugated horseradish peroxidase (Roche, Basel, Switzerland) and the BM Blue POD substrate (Roche).