Mouse ifn γ il 4 dual color elispot kit

Splenocytes isolated from PBS- or vaccines-immunized mice one week after the last immunization were subjected to ELISpot assay and IFN-γ intracellular staining. The mouse IFN-γ/IL-4 Dual-Color ELISpot kit (R&D Systems, Minneapolis, USA) was used for ELISpot assay. Briefly, 5×10⁵ splenocytes were co-incubated with 10 μg/ml NY-ESO-1 in the mouse IFN-γ-specific monoclonal antibody and IL-4-specific polyclonal antibody pre-coated microplates at 37°C for 48 h. Next, an enzyme-linked colorimetric assay was carried out for further detection. The viable cells were quantified using an ELISpot reader system (Beijing Dakewe Biotech Company, Beijing, China).
For IFN-γ intracellular staining, splenocytes were stimulated in the presence of 10 μg/ml NY-ESO-1 at 37°C for 1 h and then incubated in Golgi Plug for an additional 6 h. After staining with PerCP-anti-mouse CD4 and PE-anti-mouse CD8α (BD Pharmingen), splenocytes were fixed and permeabilized using a Cytofix/Cytoperm Kit (BD Pharmingen) according to manufacturer's instructions. The intracellular IFN-γ was detected with FITC-anti-mouse IFN-γ (BD Pharmingen) and analyzed on a BD FACS Calibur flow cytometer.

For intracellular staining of IFN-γ in CD4⁺ and CD8⁺ T cells, splenocytes were isolated one week after the last immunization, re-stimulated with NY-ESO-1 (10 μg/ml) for 1 h at 37°C, incubated for an additional 6 h in Golgi Plug and harvested, followed by staining with PerCP-anti-mouse CD4 and PE-anti-mouse CD8α. After fixation and permeabilization using a Cytofix/Cytoperm Kit, intracellular staining was achieved with FITC-anti–mouse IFN-γ (all from BD Bioscience/Pharmingen) according to the manufacturer’s instructions. After several washes, the cells were analyzed using BD FACS Calibur flow cytometer.
Splenocytes were harvested from naïve or immunized mice one week after the final immunization, and ELISPOT assays were performed using the mouse IFN-γ/IL-4 Dual-Color ELISpot kit (R & D systems, Minneapolis, USA) according to the manufacturer’s instructions. Briefly, 5 × 10⁵ splenocytes were stimulated with 10 μg/ml NY-ESO-1 in 100 μl/well RPMI 1640 supplemented with 10% FBS at 37°C for 48 h. Next, the plates were developed using an enzyme-linked colorimetric assay. The spots were quantified using an ELISPOT plate reader and software (Beijing Dakewe Biotech Company, Beijing, China).