Ionomycin

ICS assays were performed as previously described [46 (link)], with some modifications. Briefly, mouse splenocytes were added to the plate (2 × 10⁶/well) and then stimulated with the peptide pool (2 μg/mL for individual peptide) for 5 h. PMA and ionomycin (Dakewe Bioengineering, China) were used as a positive control. The cells were incubated with GolgiStop (BD Biosciences, USA) for an additional 6 h at 37°C. Then, the cells were harvested and stained with anti-CD3 (BioLegend), anti-CD4 (BioLegend), and anti-CD8α (BioLegend) surface markers. The cells were subsequently fixed and permeabilized in permeabilizing buffer (BD Biosciences, USA) and stained with anti-mouse anti-IFNγ (BioLegend), anti-TNFα (BioLegend), anti-IL-2 (BioLegend), anti-IL-4 (BioLegend), and anti-IL-10 (BioLegend) antibodies. All fluorescent lymphocytes were gated on a FACSAria flow cytometer (BD Biosciences, USA).

The ELISpot assay was performed with a mouse IFN-γ ELISpot kit (Mabtech, 3321-4APT-10) to detect the T cell response induced by the vaccine regimens. The spleens were harvested 10 days after each vaccination. Single-cell suspensions were collected after passing through a 70-μm cell strainer (Falcon, 352350). Splenocytes were then stimulated with 2 NiV F protein peptide pools (18-mers with a 10–amino acid overlap). The final concentration of each peptide was 2 μg/mL. Positive control wells were stimulated with 500 ng/mL phorbol 12-myristate 13-acetate and 10 μg/mL ionomycin (Dakewe, 2030421), whereas the negative control wells received no stimuli. The cells were then incubated with 5% CO₂ at 37°C for 24 hours. Next, a biotinylated detection antibody and streptavidin-HRP were added. Blots were developed by the addition of the substrate 3-amino-9-ethylcarbazole (AEC), which produced a colored spot after 5 minutes of room temperature incubation in the dark. Finally, IFN-γ⁺ SFCs were visualized on a Cellular Technology Ltd. imager, and counting was performed with the included ImmunoSpot software.

An ELISpot assay for the detection of IFN-γ-secreting mouse splenocytes was performed with a mouse IFN-γ ELISpot kit (Mabtech). Feshly harvested splenocytes of 5 × 10⁵ per well incubated with pools of NiV G peptides (18-mers with 10 amino acid overlap). The final concentration of each peptide was 2 μg/ml. The cells were then incubated with 5% CO2 at 37 °C. 24 h later, IFN-γ spot-forming cells were detected by staining membranes with anti-mouse IFN-γ-biotin (1 μg/ml) followed by streptavidin-ALP. Phorbol 12-myristate 13-acetate and 10 μg/ml ionomycin (Dakewe) was added to the positive-control group, whereas the negative-control group received no stimuli. Analysis was performed using the CTL ImmunoSpot Analyzer and ImmunoSpot Software (Cellular Technology). Spot-forming unit (SFU) per million cells was calculated.