Streptavidin phycoerythrin

Peripheral blood samples were obtained from healthy volunteers approved by the Clinical Research Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University (2023-0349). Informed consent from all participants was obtained. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood by density gradient centrifugation with Ficoll. T cells from PBMCs were activated with human T-cell activation beads (GenScript, L00899) in X-Vivo 15 medium at a density of 1 million/ml for 2 days and then expanded with 50 ng/ml IL-2 for several days, throughout which 2 μM GSK2879552 or vehicle was added.
The CD19-specific CAR construct containing FMC63 scFv, 4-1BB cytoplasmic domain, and CD3z cytoplasmic domain was obtained from Addgene (135992) and cloned into an EGFP-expressing lentiviral vector. Lentiviral particles were produced as described above and used to transduce pre-activated PBMCs by spin-infection. The expression of CD19-CAR was verified by flow cytometry after the sequential staining with a biotin-SP-AffiniPure F(ab)’2 fragment-specific goat anti-mouse IgG antibody (Jackson ImmunoResearch, 115-066-072) and streptavidin-phycoerythrin (BioLegend, 405203). Transduced cells were cultured and expanded in X-Vivo 15 medium supplemented with 50 ng/mL IL-2 for 6–8 days before in vitro assays or transfer into tumor-bearing NCG mice.

Human recombinant ADA (7048-AD-010, Bio-techne/ R&D Systems) at the final concentration of 5 μg/ml was incubated with 3 × 10⁵ mononuclear cells for 30 min in a CO₂ incubator. Cells were washed twice with PBS/2 mM EDTA and the percentage of ADA-bound cells was determined using biotinylated anti-ADA antibody (ab34677, Abcam) in combination with Streptavidin-Phycoerythrin (Biolegend).

Antigen-specific B cells were detected using biotinylated proteins in combination with different streptavidin-fluorophore conjugates^{42 (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.