Elisa cytokine kits

Individual spleens were collected at the end point post challenge aseptically from each mouse single cell suspensions were prepared. Splenocyte cell viability was assessed by trypan blue exclusion.³⁸ Cells were cultured in 24 well plates in triplicate at a density of 2.5 × 10⁶ cells/mL as unstimulated (control) or stimulated with 50 μg/well of Ova. Culture supernatants were collected after 72 h incubation at 37 °C and stored in aliquots at −20 °C. Concentrations of cytokines were measured in culture supernatants using ELISA cytokine kits following manufacturer’s instructions (ebiosciences, CA, USA for IL-4, IFN-γ, IL-10, IL-12, IL-17A, and TGF-β).

The levels of interferon (IFN)-γ (a classical Th1-derived cytokine) and interleukin (IL)-4 (a classical Th2-derived cytokine) in the serum of mice from different groups at week 8 were measured using commercially available ELISA cytokine kits (eBioscience, USA) as per the manufacturer's protocol.