Mouse fc block anti mouse cd16 32

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.

Single-cell suspension was generated from the spleen as described in the prior section, and from the lymph nodes by applying pressure on the tissues to pass through 40 um strainers. Single-cell suspensions were washed once with PBS, and then stained with live dead dye (fluorescent violet reactive, Thermo Fisher) diluted 333-fold in PBS at room temperature for 10 min, followed by another wash in PBS. The cells were then incubated with mouse Fc-block (anti-mouse CD16/32, BioLegend) in 1% FBS/PBS at room temperature for an additional 5 min, followed by incubation with antibody mixtures (anti-mouse GL7-FITC, anti-mouse CD4 BV510, anti-mouse CD44 AF700, anti-mouse PD1 PE-Cy7, and anti-mouse CXCR5 biotin, BioLegend, each diluted 1:200 in 1% FBS/PBS) without washing for an additional 40 min at room temperature. The cells were washed and then incubated with Streptavidin-APC at 0.5ug/mL in 1% FBS/PBS for 20 min at room temperature. The cells were then washed again and resuspended in 1% FBS/PBS for flow analysis with an LSRII 18-color instrument.