Streptavidin bv421

Fluorescent-labeled antibodies CD3-Alexa Fluor 700 (BD 557943), CD8-Alexa Fluor 700 (BD 561453), CD14-PE-Cy7 (BD 561385), CD16-PE-Cy7 (BD 560716), CD19 PerCP*Cy5.5 (BD 561295), CD20 PerCP*Cy5.5 (BD 350955), IgG-PE-Cy5 (BD 551497), IgM-BV605 (BD 562977), and IgD-BV605 (BD 563313), which target cell surface markers, were used as 1:200-fold dilution in staining. Biotin-labeled MPER peptide PDT-081 (E₆₅₄KNEQELLELDKWASLWNWFDITNWLWYIK₆₈₃-biotin) was purchased from GenScript and coupled separately to streptavidin-BV421 (BD 562426) and streptavidin-APC (allophycocyanin, BD 555335). PBMCs were stained using the LIVE/DEAD Fixable Near-IR Dead Cell Kit (Life Technologies, L34957) for 30 min on ice. Cells were then labeled with antibodies cocktail along with MPER probes for 1 h in Brilliant Staining buffer (BD 563794) on ice. Cell population CD19+/CD20+, CD3−/CD8−, CD14−/CD16−, IgG+, IgD-/IgM− MPER double positive were sorted using BD FACSAria III sorter into individual wells of a 96-well plate containing lysis buffer and plates were immediately sealed and stored at −80 °C. The single B-cell sorting strategy is shown in the Supplementary Fig. 9.

Lymph node cell suspensions from influenza infected ferret were stained with the following panel: live/dead Blue (Thermo Fisher), CD79a PerCP-Cy5.5 (HM47; BioLegend)^{24 (link)}, CD8 AF700 (OKT8; Thermo)^{24 (link)}, CD4 FITC (from CSIRO)^{25 (link)}, BCL6 AF647 (K112-91; BD), BCL6 AF647 (IG191E/A8; BioLegend), CXCR5 BV421 (L138D7; BioLegend), CXCR5 BB515 (RF8B2; BD), CXCR5 PE (2G8, BD); CXCR5 Biotin (in-house), Streptavidin BV421 (BD), PD-1 BV786 (29F.1A12; BioLegend), PD-1 BV421 (EH12.2H7; BioLegend), PD-1 PE (in-house). For BCL6 staining, cells were fixed, permeabilized, and stained using the BD Transcription Factor Buffer kit (BD) according to the manufacturer’s instructions. Macaque LN suspensions were stained with Live/dead Aqua (Thermo Fisher), CD4 BV605 (L200; BD), CXCR5 PECy7 (MU5UBEE, Thermo Fisher), and CD3 BUV737 (SP34-2, BD). All samples were acquired on a BD LSR Fortessa using BD FACS Diva and data was analyzed in FlowJo v10.

Single-cell suspensions were prepared from splenocytes. To stain dead cells, the suspensions were incubated with fixable efluor 780 (Affymatrix, Santa Clara, CA) diluted at 1:1,000 dilution in PBS for 10 min at room temperature. Cells were washed and stained using FACS buffer containing 2% FBS, 0.5M EDTA in PBS. The following antibodies were used for surface staining at room temperature: α-CD4 (BD Biosciences, 1:200, GK1.55), α-PD-1 (BD Biosciences, 29F.1A12), α-CXCR5 (biotin, BD Biosciences, 2G8; BioLegend, L138D7), α-GL7 (BD Biosciences, GL-7), α-FAS (BD Biosciences, J02), α-CD25 (BioLegend, San Diego, CA, PC61), α-IL-6Rα (biotin, Biolegend, D7715A7), GP130 (R&D system, Q6PDI9), α-IL-21R (biotin, eBioscience, eBioA9), α-ICOSL (biotin, HK5.3, BioLegend), CD19 (6D5, Biolegend), CD23 (B3B4, eBioscience), Bcl6 (7D1, Biologend). To detect biotinylated CXCR5, IL-6Rα, IL-21R, and ICOSL antibodies, cells were further incubated with streptavidin-BV-421 (BD Bioscience, 1:500) for 15 min at room temperature. For intracellular staining, samples were fixed with the Foxp3 Fix/Perm buffer set by following the manufacturer's instructions (eBioscience). Samples were then intracellularly stained with α-Foxp3 (BioLegend, 150D, 1:100) antibody. Flow cytometry data were acquired on LSRII flow cytometer (BD Biosciences) and analyzed using the FlowJo software v10 (Tree Star, Inc., Ashland, OR).

TTd protein (Statens Serum Institut, DK) was conjugated to biotin via amide linkage following reaction of TTd with N-hydroxysuccinimide (NHS) biotin ester (EZ-link sulfo-NHS-LC-biotin labelling kit, ThermoFisher Scientific, Rockford, IL). Excess free biotin was removed with size-exclusion chromatography using Zeba™ 7 kDa molecular weight cut-off desalting columns (ThermoFisher Scientific). The degree of biotinylation was determined to be 6 biotin molecules per molecule of TTd, as measured with the 4’-hydroxyazobenzene-2-carboxylic acid/Avidin biotin quantification kit (ThermoFisher Scientific). Tetramers were created by reacting biotin-TTd with either streptavidin-BV421 (BD Biosciences) or streptavidin-AF647 (BioLegend) at 4:1 molar ratios for 20 mins, on ice, and centrifuged at maximum speed on a benchtop microcentrifuge (18,000 x g) for 10 mins (4°C) to remove aggregates.

Bone marrow plugs recovered from tibiae and femurs of mice (n = 6–9) from each experimental group were incubated in digestion buffer containing 500 µg/mL LiberaseDL^® at 37 °C in three intervals of 15 min to release SSPCs. Marrow cells were filtered using a cell strainer (40 µM) to remove debris and then stained with propidium iodide to assess viability. Cells were also stained with antibodies against the lineage markers TER-119 (Tonbo Biosciences, Cat #: 35–5921, clone #: TER-119), CD31 (BD Pharmingen, Cat #: 553372, clone #: MEC13.3) and CD45 (Tonbo Biosciences, Cat #: 35–0451, clone #: 30-F11), biotinylated anti-LEPR antibody (R&D Systems, Cat # BAF497) and BV421 Streptavidin (BD Biosciences, Cat #: 563259). Thereafter, the Lin⁻/LEPR⁺ fraction was sorted using a BD FACSAria3 flow cytometer in the UF Scripps Biomedical Research Flow Cytometry Core using a gating strategy that excludes doublets and non-viable cells.