Ccl22 mdc

CCL17 and CCL22 levels in cell culture supernatants and sera were quantified using the ELISA kits, Quantikine Human CCL17/TARC, and CCL22/MDC (R&D Systems), respectively. Cell lines (2.5 × 10⁵/mL) were cultured in 96-well round-bottomed plates, and supernatants were collected after 24, 48, and 72 h. All sera were taken at diagnosis and were frozen at −80 °C. The capture antibody was coated onto the bottom of supplied 96-well ELISA plates and was incubated overnight at 4 °C. Cell culture supernatants (100 µL) or sera (50 µL) were added to each well. The plates were washed after 2 h of incubation at room temperature, and biotinylated anti-chemokine antibody (detection antibody) was added to each well. After 2 h, the plates were washed again and streptavidin-labeled horseradish peroxidase (1:200) was added for 20 min. A substrate solution was added after washing and incubated for 20 min in the dark, and the reaction was stopped by adding 2 N H₂SO₄. The absorbance of each well was determined at 450 nm using a microplate reader. For cell culture supernatants, measurements were done in triplicate. The results correspond to means ± SD.