Splenocytes from immunized animals were isolated at 14 days following the last immunization and assessed by ELISpot for IFN-γ, IL-4, and IL-17A production. Multiscreen plates, 96 wells (MAHA, MAIP, or MSIP, Millipore), were coated overnight at 4°C with anti-mouse IFN-γ (5 μg/ml; AN18, Mabtech), anti-mouse IL-4 (5 μg/ml; BVD4-1d11, BD Biosciences), or IL-17A (5 μg/ml; Mabtech). Plates were washed five times with PBS and blocked with RPMI 1640 (Gibco, Life technologies) containing 10% FBS [supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), 2 mM l-glutamine, 1 M Hepes, and 0.1 mM 2-mercaptoethanol] for 2 hours at 37°C. Splenocytes were added at a final concentration of 0.5 × 106 per well and coincubated with recall antigens (1, 5, and PADRE conjugated to an unrelated peptide at a final concentration of 25 μg/ml) in complete media for 12 to 16 hours at 37°C (IFN-γ), 20 to 24 hours (IL-4), and 40 to 48 hours (IL-17A). Media alone and positive control concanavalin A (1 μg/ml final) wells were also added. Following incubation, plates were washed with PBS, and anti-mouse IFN-γ–biotin (1 μg/ml; R4-6A2, Mabtech), anti-mouse IL-4–biotin (BVD6-24G2, Mabtech or BD Biosciences), or anti-mouse IL-17A–biotin (BD Biosciences) in 0.5% FBS/PBS was added for 2 hours at room temperature (IFN-γ and IL-17A) or overnight at 4°C for IL-4. Plates were washed, and streptavidin-ALP (1 μg/ml) or ExtrAvidin-ALP (1 μg/ml) in 0.5% FBS/PBS was added for a further 1.5 hours at room temperature. Plates were given a final wash with PBS followed by reverse osmosis water, and spots were developed using an AP colorimetric kit (Bio-Rad). Once plates were dry, spots were counted using an AID ELISpot Reader system.

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