Streptavidin pe

(1) Construction of CD137L-expressing cell lines. TrueORF cDNA expression clones for C-terminal Myc- and DDK (Flag)–tagged M. musculus TNFSF9 were obtained (cat. # MR220933, Origen). Circular plasmid DNA was isolated and restricted with ApaLI (NEB) to linearize the vector DNA. Linearized DNA was then transfected into HEK293 cells using Lipofectamine 2000 and stable integrants selected for using 400–500 μg/ml of Geneticin (G418). After isolation and expansion of individual cell clones, high mouse CD137L-Myc-DDK expressers were stained with rat anti-mouse CD137L antibody (clone: TKS-1, BioLegend) or Rat IgG2a isotype control antibody (clone: RTK2758, BioLegend), followed by biotinylated mouse anti-Rat IgG2a antibody (clone: RG7/1.30, BD Biosciences) and streptavidin PE (BioLegend), identified using BD FACSCanto (BD Biosciences).
(2) Confirmation of specific binding of sCD137 to CD137L. HEK-mCD137L or control HEK293 cells (2 × 10⁶) were preincubated with 1 ug/sample of anti-CD137L Ab (TKS-1) or the same volume of PBS for 30 min at 4°C. After washing cells to remove excess CD137L antibody, cells were incubated with titrated doses of sCD137 for 30 min at 4°C. Then cells were stained with biotinylated rat anti-mouse CD137 antibodies (clone: 17B5, BioLegend) followed by streptavidin PE (BioLegend). The level of bound CD137 was analyzed using FACSCanto (BD Bioscience).

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.

Mice were immunized subcutaneously at the tail base, and inguinal LNs were collected on day 14 post immunization. For GC analysis, cells were mechanically digested and strained through 70 µm filters prior to staining for GC B cell analysis, cells were stained for viability (Live/Dead Fixable Aqua, Thermo Fisher), CD3e (dye: BV711, clone 145‐2C11; BioLegend), B220 (dye: PE‐Cy7, clone RA3‐6B2; BioLegend), CD38 (dye: FITC, clone 90; BioLegend), and GL7 (dye: PerCP‐Cy5.5, clone GL7; BioLegend), and for antigen‐specific staining biotinylated eOD conjugated to streptavidin‐BV421 and streptavidin‐PE (BioLegend). For T_FH analysis, cells were stained for viability (Live/Dead Fixable Aqua, Thermo Fisher) and against B220 (dye: BV510, clone RA3‐6B2; BioLegend), CD4 (dye: FITC, clone GK1.5; BioLegend), CD44 (dye: PE‐Cy7, clone IM7; BioLegend), PD‐1 (dye: BV421, clone RMP1‐30; BD Biosciences), and CXCR5 (dye: PE, clone 2G8; BD Biosciences). Samples were analyzed by flow cytometry on a BD Celesta and analyzed on FlowJo.