Regulatory T cell and Memory B cell Staining

Regulatory T cell staining was performed as previously described [20 (link)]. Briefly, 10⁶ splenocytes/well were seeded in 96-well U-bottom plates and stained with anti-mouse CD4 BV650 (RM4-5, Biolegend, San Diego, CA, USA), anti-mouse CD25 APC (PC61, BD Pharmingen, San Jose, CA, USA), and Fixable Viability Dye eFluor450 (eBioscience, San Diego, CA, USA). Cells were fixed, permeabilized and stained intracellularly with anti-mouse Foxp3 PE (MF14, Biolegend, San Diego, CA, USA). To measure samples, FACS-LSR II (BD, Franklin Lakes, NJ, USA) was used and data analyzed using FlowJo (Tree Star, Ashland, OR, USA).
For memory B cell staining, 10⁶ splenocytes/well were seeded in 96-well U-bottom plates and surface stained with anti-mouse CD19 Qdot655 (6D5, Thermo Fisher Scientific, Schwerte, Germany), anti-mouse CD80 APC (16-10A1, BD, Franklin Lakes, NJ, USA), anti-mouse IgD PE (217–170, BD, Franklin Lakes, NJ, USA) and Fixable Viability Dye eFluor450 (eBioscience, San Diego, CA, USA). HA-specific B cells were detected by surface staining with soluble HA of the influenza A virus (strain A/Puerto Rico/8/1934/H1N1) labeled with Alexa488 using the Alexa488 Protein Labeling Kit (Thermo Fisher, Schwerte, Germany).