3 amino 9 ethylcarbazole aec substrate solution

IFN-γ production was detected using an IFN-γ Chicken antibody pair kit (life technologies). In brief, MAIPS4510 MultiScreenTM-IP 96 well plates (Millipore, UK) were coated overnight at 4°C with 2μg/ml mouse anti-ChIFN-γ in PBS. Plates were washed twice with blocking buffer (RPMI 1640 plus 2% FCS) and incubated (1 hour; 37°C, 5% CO₂) with blocking buffer. Mononuclear cells were seeded in triplicates at 5.0 x10⁵ cells per well, stimulated with medium alone or in the presence of PMA (50 ng/ml) plus Ionomycin (Ion; 1 μg/ml); (Sigma, UK) and incubated (41°C, 5% CO₂) overnight. Plates were washed twice with SQ water followed by three time wash with washing buffer (PBS + 0.1% Tween 20). 1μg/ml of anti-chicken IFN- γ biotinylated antibody in assay buffer (PBS + 0.1% Tween 20 + 1.0% BSA) was added to the plate and incubated for 1 hour in the dark at RT. Plates were washed with washing buffer and incubated for a further 1 hour with Streptavidin-HRP (1/1250) in assay buffer. A final wash with washing buffer was performed and a 3-Amino-9-ethylcarbazole (AEC) substrate solution (BD Biosciences, UK) was used to develop the colour at RT in the dark. After 20 min, the reaction was inactivated by washing in distilled water, air dried overnight and spots forming units (SFU) were counted using an automated ELISPOT reader.