Splenocyte Cytokine Profiling for RBD Peptides

Splenocytes at 1 × 10⁶ cells/well were exposed to an RBD peptide pool (2 μg/mL for each peptide) or 5 μg/mL of each peptide in the presence of a protein transport inhibitor containing brefeldin A (BD Biosciences) for 12 h at 37 °C. Intracellular cytokine staining was performed according to a protocol modified from a previous paper [8 (link)]. The treated cells were incubated with LIVE/DEAD Fixable Red Dead Cell stain (Invitrogen) for 20 min at room temperature. The cells were washed, incubated with an anti-CD16/32 antibody (2.4G2) solution to block the Fc receptor, and stained for surface markers (CD3ε, CD4, and CD8 molecules), followed by intracellular IFN-γ cytokine staining using the Foxp3/transcription factor buffer set (eBioscience). The antibodies used were anti-CD3ε BUV737 (17A2), anti-CD4 APC (RM4–5), anti-CD8α PE-Cy7 (53–6.7), and anti-IFN-γ BV650 (XMG1.2). All the antibodies were obtained from BD Biosciences. The stained cells were acquired on a FACS Aria Fusion (BD Biosciences) and were analyzed via FlowJo^TM V10 (TreeStar, Ashland, OR, USA).