Percp anti mouse cd4

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.

Sixty-three mice were randomly divided into nine groups (seven mice in each). Five groups were immunized i.v. with LMΔ-msv, LIΔ-msv, LMΔ-lacZ, LIΔ-lacZ, and NS. The other four groups were prime-boost immunized i.v. with LMΔ-msv → LMΔ-msv, LMΔ-msv → LIΔ-msv, LIΔ-msv → LMΔ-msv, and LIΔ-msv → LIΔ-msv, respectively. Boost immunization was carried out 40 days after prime immunization. Immunization doses were 0.1-fold of the 50% lethal doses of each strain. Splenocytes were harvested from the immunized mice 9 days after the last immunization. Isolated splenocytes were stimulated with the peptide pool listed in Table 2 at the final concentration of 1 μg/mL prepared in 10 μg/mL Golgi stop (BD PharMingenTM, USA) or no stimulant in 96-well plates at 37°C for 5 h, and then processed for flow cytometry analysis as described previously (16 (link)). After centrifuging for 5 min at 1, 300 × g, the cells were then washed and co-stained with FITC-Anti-Mouse CD3 (BD, USA), PerCP-Anti-Mouse CD4 (BD, USA), and APC-CY7-Anti-Mouse CD8 (BD, USA) at 4°C for 30 min. The surface-stained cells were washed and permeabilized using Cytofix/Cytoperm kit (BD, USA). The cells were then washed and stained for 45 min on 4°C for PE-Anti-Mouse IFN-γ (Biolegend, USA), PE-Cy7-Anti-Mouse TNF-α (Biolegend, USA), and APC-Anti-Mouse IL-2 (Biolegend, USA). The stained cells were analyzed using flojow 10.0.

Single cell suspensions were stained for 20 min at 4°C with the following fluorochrome-conjugated monoclonal antibodies or tetramers in FACS buffer (PBS with 5% FBS, 0.1% sodium azide): PE anti-mouse αGalCer-loaded CD1d tetramers (PBS57/Dimerix from the NIH Tetramer Facility, Washington, DC, USA), FITC anti-mouse TCRβ, PerCP anti-mouse CD4, Pacific Blue CD8, APC-Cy7 anti-mouse CD3 (BD Biosciences, San Diego, CA, USA). For intracellular cytokine staining, single-cell suspensions were stimulated for 2.5 h with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 µg/mL ionomycin (both from Sigma-Aldrich, St. Louis, MO, USA) in the presence of 10 µg/mL Brefeldin A (Sandoz, Princeton, NJ, USA). Cells were collected and labeled for iNKT cell surface markers and then fixed and permeabilized with fixation and permeabilization buffer (BD Biosciences, San Diego, CA, USA) and stained with PE-Cy7 anti-mouse IL-17 and FITC anti-mouse IFN-γ mAbs (BD Biosciences, San Diego, CA, USA). Dead cells were stained with AmCyan-conjugated fixable viability dye (eBioscience, San Diego, CA, USA) and excluded from the analysis. Flow cytometry data were acquired on a FACSCanto II and analyzed with FACS Diva software (BD Biosciences, San Diego, CA, USA).