Intracellular Cytokine Staining for Virus-Specific CD8+ T Cells

ICS was performed using the Cytofix/cytoperm kit (BD Pharmingen, San Diego, CA, USA) according to the manufacturer’s instructions. A total of 1 × 10⁶ freshly isolated splenocytes were stimulated with 1 μM of an individual peptide or inactivated virus in the presence of 1 μg/mL brefeldin A (Sigma-Aldrich, Dorset, UK) in 1000 μL of culture medium at 37 °C for five hours [12 (link)]. Cultures were stimulated with UV-inactivated RSV Long strain, F, NP, and M2-1 peptides as shown in Table 1. F peptides are recognition sites of palivizumab (synagis®). NP_306–314 is based on the human experiments and the others are on mouse. After the stimulation, splenocytes were washed and incubated with an anti-CD8 antibody (R&D Systems, Minneapolis, MN, USA) at 4 °C for 30 min. Splenocytes were fixed with fixation buffer in the Cytofix/cytoperm kit, and intracellular cytokines were stained with a goat IgG antibody against IFN-γ (R&D Systems, USA) and anti-goat IgG PE-Cy7 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4 °C for 60 min. Cellular populations were analyzed using flow cytometry with Cytomics FC 500 (Beckman Coulter, Inc., Indianapolis, IN, USA) and counted until 100,000 cells.