Generating FITC and PE Labeled MD39 Tetramers

To generate FITC and PE labelled MD39 tetramers, biotinylated avi-tagged MD39 produced as previously mentioned were incubated with molar ratios of Streptavidin-FITC or Streptavidin-PE (BioLegend) for 30 min at room temperature^{8 (link),56} . Single-cell suspensions from spleen or lymph nodes of the animals were first washed with PBS, then stained with live dead dye (fluorescent violet reactive, Thermo Fisher) diluted in PBS at room temperature for 10 min, followed by the addition of mouse Fc-block (anti-mouse CD16/32, BioLegend) diluted 1:100 in 1% FBS/PBS and incubation at room temperature for additional 30 min. The cells were washed, and then incubated with 5ug/mL Streptavidin-MD39-FITC and Streptavidin-MD39-PE at room temperature for 45 min. Antibody mixtures (anti-mouse CD19-PE-Cy7, Anti-mouse IgD APC-Cy7, and Anti-mouse IgM BV711, BioLegend, each diluted 1:200 in 1% FBS/PBS) were then added to the cells without any intermediate washing steps, followed by incubation for another 45 min at room temperature. The cells were washed once with 1% FBS/PBS, and then resuspended at 10 million/mL concentration in 1% FBS/PBS for sorting with the FACS ARIA II instrument. CD19 + IgM− IgD− MD39-Tetramer-FITC + MD39-Tetramer-PE + cells were then sorted in bulk into 1.5 mL Eppendorf tube containing 1% FBS/PBS for downstream cDNA prep with 10× genomics.