Facsaria 3

Spleens or lymph nodes (inguinal and cervical) were dissociated on a nylon mesh. Cell suspensions were treated with red blood cells (RBC) lysis buffer (0.83% NH₄Cl vol/vol). Blood was harvested into heparin tubes and RBC lysed. Cell suspensions were incubated with 2.4G2 Fc Block and stained with fluorescently tagged antibodies (See Supplementary Table 1) in FACS buffer (PBS, 1%FCS, 2 mM EDTA, 0.02% sodium azide). Brilliant stain buffer (BD) was used when more than two Abs were conjugated with BD Horizon Brilliant fluorescent polymer dyes. Ova_257–264/K^b, gB_498–505/K^b, GP_33–41/D^b, GP_276–284/D^b, LLO_91–99/K^d and p60_217–225/K^d biotinylated monomers (1 mg/mL) obtained from the NIH tetramer Core Facility, were conjugated with PE-labeled Streptavidin (1 mg/mL) as follow: 6.4 μL of PE-Streptavidin were added to 10 μL of monomers every 15 min 4 times on ice. Newly generated tetramers (1/400–1/500 dilution) were used to stain cells for 1 h at 4 °C. For transcription factor (TF) intracellular staining, cells were fixed and stained according to the eBioscience Foxp3 Transcription Factor Staining Buffer Set protocol. Data acquisition was done using BD LSR II, FACSAria III or Cytek Aurora flow cytometer. All flow cytometry data were analyzed using FlowJo v9 or v10 software (TreeStar).