2.7.2. For Influenza Study

Spleens were isolated from BALB/c mice 14 days post vaccination. Single-cell suspensions of splenocytes were made by homogenizing and processing the spleens through a 40-μm cell strainer. Cells were then re-suspended in ACK Lysing buffer (GibcoTM, Gaithersburg, MD, USA) for 5 min to lyse red blood cells before two washes with PBS and final re-suspension in RPMI complete media (RPMI 1640 + 10% FBS + 1% penicillin–streptomycin). Two hundred thousand splenocytes were added to each well and stimulated overnight at 37 ◦C in 5% CO2 with R10 (negative control), concanavalin A (3 μg/mL; positive control), or 15 mer influenza hemagglutinin peptides overlapping by 11 amino acids spanning the length of the consensus HA1 hemagglutinin protein (GenScript). The HA1 peptides were pooled at a concentration of 1 mg/mL/peptide into four pools as antigens for specific stimulation of IFN-γ release.
