Fitc anti mouse ifn γ

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.

Splenic CD4⁺ cells from approximately 28-day-old mice were obtained using the CD4⁺ T cell isolation kit (Miltenyi Biotec, 130-104-454) or by sorting with the BD FACSAria II cell sorter. CD4⁺ cells were processed using the Mouse Th1/Th2/Th17 (BD Biosciences, 560758) and Th17/Treg (BD Biosciences, 560767) phenotyping kits. Cells were cultured for 3–4 hours with or without 50 ng/mL PMA and 1 μg/mL ionomycin and then harvested for RNA-Seq or flow analysis. The following antibodies were used: PerCP-Cy5.5 anti–mouse CD4 (BD Biosciences, 550954), PE anti–mouse IL-17a (BD Biosciences, 559502), FITC anti–mouse IFN-γ (BD Biosciences, 554411), APC anti–mouse IL-4 (BD Biosciences, 554436), and Alexa Fluor 647 anti–mouse FOXP3 (BD Biosciences, 560402).

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).

Freshly isolated splenocytes or pulmonary immune cells were resuspended at a concentration of 1 × 10⁷cells/mL. Cells were stimulated with 10 μg/mL antigens (LppZ and Ag85A) or tuberculin PPD for 20 h or treated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 500 ng/mL Ionomycin (both from Sigma Aldrich) for 5 h. GolgiStop solution (BD Biosciences) was added to cell culture during the last 5 h of ex vivo stimulation. For surface staining, cells were incubated with PE-Cy5-anti-mouse CD3 (eBioscience), Pacific Blue-anti-mouse CD4 (Biolegend), APC-Cy7-anti-mouse CD8 antibodies, biotin-Annexin V and PerCP-streptavidin (BD Biosciences) for 30 min in the dark at 4°C. After washing with PBS containing 2% FBS, cells were fixed and permeated by the Cytofix/Cytoperm reagent from Fixation/Permeabilization kit (BD Biosciences). Cells were incubated with FITC-anti-mouse IFN-γ, PE-anti-mouse IL-2 (both from BD Biosciences), and APC-anti-mouse TNF-α (Biolegend) antibodies for 45 min at 4°C. Cells were washed, resuspended in PBS and acquired with a FACS CantoII flow cytometer (BD Bioscience) in 2 h. Data were analyzed by using FlowJo software 7.5 (Treestar Inc.).

Cells were stained with APC-anti-mouse CD3e (553066; BD Pharmingen, San Diego, CA) and PE-anti-mouse CD4 (557308; BD Pharmingen) for 30 min at 4 °C, followed by washing twice with PBS containing 1% FBS and 0.03% sodium azide. For intracellular staining, cells were fixed and permeabilized using eBioscience™ Intracellular Fixation & Permeabilization Buffer (Thermo Fisher Scientific) followed by staining with FITC-anti-mouse IFN-γ (554411; BD Pharmingen). After washing twice with PBS containing 1% FBS and 0.03% sodium azide, the cells were analyzed by FACScalibur (Becton, Dickinson and Company, Franklin Lakes, NJ) using FlowJo software (Becton Dickinson).