Elisa kits for il 4

Twenty-four hours following the last OVA challenge, the mice were sacrificed by pentobarbital overdose (50 mg/kg body weight) and tracheotomy was done. Ice-cold PBS (0.5 ml) was infused into a lung and BALF was collected via tracheal cannulation. BALF was collected by three successive aspirations (total volume 1.5 ml) [30 (link)] and then centrifuged (4℃, 250 g) for 5 min. The supernatant collected was stored (−70℃) until use. The supernatant was used for assessment of cytokines.
Cytokines IL-4, IL-5, IL-6, IL-13, IL-17A and IFN-γ in the BALF were determined by ELISA. The kits for analysis of IL-6, IL-17A and IFN-γ were purchased from R&D Systems (Minneapolis, MN, USA). ELISA kits for IL-4, IL-5 and IL-13 were purchased from Biolegend (San Diego, CA, USA). The analysis was performed as per manufacturer's protocol. Further, for determination of differential cell counts, cell pellets were re-suspended in PBS and stained with Wright–Giemsa stain (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).