Splenocyte Isolation and Flow Cytometry

Spleen cells were isolated at 14 days post infection (dpi). Homogenized single-cell suspensions were prepared in 6 mL of DMEM (Capricorn Scientific, Ebsdorfergrund, Hessen, Germany) supplemented with 10% FBS (Atlas Biologicals, Fort Collins, CO, USA) and 1% penicillin/streptomycin using gentleMACS™ Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were passed through a 70-µm cell strainer (SPL Life Sciences, Gyeonggi-do, South Korea), and centrifuged at 314× g for 7 min at 4 °C, followed by re-suspension and incubation in RBC lysis buffer (BioLegend, San Diego, CA, USA) at 4 °C for 5 min. After washing (314× g 7 min at 4 °C), cells were kept on ice in FACSFlow sheath fluid (BD Biosciences, San Jose, CA, USA) containing 0.05% FBS (Atlas Biologicals, USA) and Fc block (CD16/CD32 Fcγ III/II, BioLegend, San Diego, CA, USA) (1/1000 dilution) for 30 min in the dark at 4 °C. Next, 10⁵ cells per sample were incubated for 30 min in the dark at 4 °C, with antibody cocktails specific for different splenocytes populations, followed by flow cytometry analysis using a BD Accuri™ C6 Plus flow cytometer (BD Biosciences, San Jose, CA, USA). The gating strategies used have previously been published [84 (link)], and the percentage of each population was determined by dividing the number of events within a specific gate, by the total number of events within live gate.