Antigen-specific B cells were detected using biotinylated proteins in combination with different streptavidin-fluorophore conjugates42 (link). Biotinylated proteins were multimerized with fluorescently labeled streptavidin for 1 h at 4 °C. Full-length spike protein (R&D Systems) was mixed with streptavidin-Brilliant Violet 421 (BioLegend) at a 10:1 mass ratio (for example, 200 ng spike with 20 ng streptavidin; approximately 4:1 molar ratio). Spike RBD (R&D Systems) was mixed with streptavidin allophycocyanin (BioLegend) at a 2:1 mass ratio (for example, 25 ng RBD with 12.5 ng streptavidin; approximately 4:1 molar ratio). Biotinylated influenza hemagglutinin pools (A/Brisbane/02/2018/H1N1, B/Colorado/06/2017; Immune Technology) were mixed with streptavidin-phycoerythrin (BioLegend) at a 6.25:1 mass ratio (for example, 100 ng hemagglutinin pool with 16 ng streptavidin; approximately 6:1 molar ratio). Streptavidin-Brilliant Violet 711 (BD Biosciences) was used as a decoy probe without biotinylated protein to gate out cells that nonspecifically bind streptavidin. Antigen probes for spike, RBD and hemagglutinin were prepared individually and mixed together after multimerization with 5 µM of free D-biotin (Avidity LLC) to minimize potential cross-reactivity between probes.
Free d biotin
Manufactured by Avidity
Free D-biotin is a pure form of the essential vitamin biotin. It is a colorless, odorless crystalline solid that is soluble in water and organic solvents. Biotin is a cofactor for several enzymes involved in carboxylation reactions and plays a role in metabolism.
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2 protocols using free d biotin
1
Multimerized Antigen Probes for B Cell Detection
2
Antigen-Specific B Cell Detection
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Antigen-specific B cells were detected using tetramer-probes. Biotinylated spike and RBD proteins (R&D Systems) were multimerized with fluorescently labeled streptavidins (SAs) for 1 hour at 4 degrees Celsius. Full-length spike protein was mixed with SA-BV421, using ~100 ng of spike and ~20 ng of SA for each sample (~4:1 molar ratio). RBD was mixed with SA-AF647, using ~25 ng of RBD and ~12.5 ng of SA (~4:1 molar ratio). The SA-PerCP was used as a decoy probe. After 1 hour, all tetramers were mixed in free D-biotin (5 μM, Avidity LLC) to minimize cross-reactivity. PBMCs were incubated for 1 hour at 4 degrees Celsius with the mix of tetramers, washed and stained with surface markers, and labeled for viability with L/D Zombie NIR (1:500) for ~10 min prior to being acquired with the 5L Aurora (Cytek).
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