Mouse il 12 il 23 total p40 elisa ready set go

The suspension of spleen cells from immunized mice was prepared by mechanical disruption. Ammonium chloride lysing buffer was used for the lysis of red blood cells. The prepared cells were seeded into 96-well cell culture plates at a density of 1 × 10⁶ cells/mL and stimulated either with VLPs (HaVP1-PADRE-4, TSPyV VP1 and HaPyV VP1) at a concentration 5 μg/mL or polyclonal activators Concavalin A (ConA) (Sigma-Aldrich) and lipopolysaccharide (LPS) (Sigma-Aldrich) added at concentrations 3 μg/mL and 1 μg/mL, respectively, to the growth medium: RPMI-1640 containing 10% FBS (Merck-Millipore) and antibiotics. After 24 h and 48 h of incubation, culture supernatants were collected and stored at −80 °C until analysis.
Interleukin 12 (IL-12), interleukin 2 (IL-2) and interferon gamma (IFN-γ) concentrations in mouse spleen cell cultures were determined by sandwich ELISA using Mouse IL-12/IL-23 total p40 ELISA Ready-Set-Go, Mouse IFN gamma ELISA Ready-Set-Go (eBioscience, San Diego, CA, USA) and Mouse IL-2 ELISA set (BD Biosciences, San Jose, CA, USA) according to the recommendations of the suppliers.

The colon from mice was collected, carefully washed with saline, and protein extracted using Lysis Buffer (Tris–HCl 50 mM, NaCl 150 mM, EDTA 5 mM, and Triton-X100 1%) and Cocktail Protease Inhibitor (04,693,159,001, Roche). The expression of IL-12, TNF-α, IFN-γ, IL-1β, IL-10, and IL-33 in the colon were assayed by ELISA according to the manufacturer’s instructions (Mouse TNF-α ELISA Ready-Set-Go, eBioscience, 88–7324–88; Mouse IL-1β ELISA Ready-SET-Go, eBioscience, 88–7013–88; Mouse IL-12/IL-23 total p40 ELISA Ready-SET-Go, eBioscience, 88–7120–88; Mouse IL-10 ELISA Ready-SET-Go 2° Generation, eBioscience, 88–7105–88; Mouse IL-33 DuoSet ELISA kit, R&D Systems, DY3626; Mouse IFN-gamma DuoSet ELISA kit, R&D Systems, DY485).