Splenocyte proliferation assay

Spleens of vaccinated mice (S1, L1 or L2; 100 µg) were harvested 2 weeks after the last injection and single-cell suspension was made. The cells were stained with 5 µm CFSE (CarboxyFluoroscein Succinimidyl Ester, Invitrogen:{"type":"entrez-nucleotide","attrs":{"text":"C34554","term_id":"2370695","term_text":"C34554"}}C34554). Briefly, cells were incubated in the dark with the dye for 8–10 min. The cells were then washed two times with 10 mL ice-cold PBS+10% FBS (fetal bovine serum) to get rid of excess dye. The cells were then plated along with matched antigen (S1, L1 or L2; 1 µg/well) for 4 days. Similarly for controls, CFSE-stained splenocytes from naïve mice were also plated with all three peptides in culture. On day 5, cells were collected and stained with surface markers for T cells. The samples were run using BD Fortessa and analyzed on BD FlowJo and ModFit.