Pe cy7 anti mouse il 2

Splenocytes and PBMCs were prepared from mice and NHPs respectively. Splenocytes or PBMCs (1 × 10⁶) were seeded into 96-well round bottom plates (Corning, Cat. 3799) and stimulated with 1% DMSO as negative control, 1 μg/mL/peptide of a peptide pool from SARS-CoV-2 spike protein (synthesized by GenScript, China) or 100 ng/mL PMA & 1 μg/mL Ionomycin as positive control. 1 μg/mL Brefeldin A (BD, Cat. 555029) was added to inhibit cytokine secretion. After incubation at 37 °C 5% CO₂ for approximately 6 h, cells were harvested and stained with LIVE/DEAD Aqua and a panel of flow cytometry antibodies specific for cell surface markers: PB anti-mouse CD3 (BioLegend, Cat. 100214), FITC anti-mouse CD4 (BD, Cat. 553047) for mouse study or PB anti-human CD3 (BD, Cat. 558124), FITC anti-human CD4 (BD, Cat. 550628) for NHP study at 2–8 °C for 30 min. Following washing and permeabilization (BD, Cat. 554714), cells were further stained with flow cytometry antibody mixture: APC anti-mouse IFN-γ (BD, Cat. 554413), PE-Cy7 anti-mouse IL-2 (BD, Cat. 560538), PE anti-mouse TNF-α (BD, Cat. 554419) for mouse study or APC anti-human IFN-γ (BD, Cat. 554702), PE-Cy7 anti- human IL-2 (BD, Cat. 560707), PE anti- human TNF-α (BD, Cat. 557068) for NHP study at 2–8 °C for 30 min. The stained cells were analyzed by BD FACSCantoII flow cytometry.

Spleens were isolated from immunized C57BL/6 mice 3 days after the last DC administration (n=3). Prepared splenocytes (1×10⁶ cells/well) were restimulated in 24-well plates with 5 µg/mL freshly-prepared vaccine protein for 6 h in the presence of recombinant mouse IL-2 (20ng/ml; PeproTech). 50ng/mL PMA, 1ug/ml Ionomycin and 10 µg/mL Brefeldin A (Absin) was added to accumulate intracellular cytokines. After restimulation, the cells were firstly incubated with anti-mouse CD3, CD4 and CD8 antibodies for surface staining. Subsequently, intracellular staining for IL-2, IFN-γ, Granzyme B and Perforin were performed after these cells were fixed and permeabilized. Fixable viability dye was used to gate out dead cells. Data were collected on FACS verse (BD Biosciences) and analyzed with Flow Jo software. The antibodies used in this study were including FITC anti-mouse CD3 (Thermo Fisher), PE anti-mouse CD4 (BD Biosciences), PerCP-Cy™5.5 anti-mouse CD8a (BD Biosciences), PE-Cy7 anti-mouse IL-2 (BD Biosciences); BV510 anti-mouse IFN-γ (Biolegend); BV421 anti-mouse Granzyme B (Biolegend); APC anti-mouse Perforin (Biolegend); Fixable Viability Stain 780 (BD Biosciences); Fixation/Permeabilization Kit (BD Biosciences).