Quantification of IFN-γ secretion in T cell-APC co-cultures

T cell clones (2 x 10⁴) were co-cultured with peritoneal exudate cells (2.5 x 10⁴) as antigen presenting cells and synthetic peptides (CHI Scientific, Genescript) or whole porcine insulin (Sigma-Aldrich) for 48 hrs in 0.25 ml in 96-well plates. Interferon gamma (IFN-γ) secretion in the culture supernatant was measured via ELISA using anti-mouse IFN-γ (BD Pharmingen) to coat ELISA plates, biotin-labeled anti-mouse IFN-γ (BD Pharmingen), streptavidin-peroxidase (Sigma), and 2,2’-Azino-bis(3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt for colorometric detection. Absorption was quantified at 415 nm on a microplate reader (iMARK, BIORAD) (46 (link), 47 (link)).