Af647 affinipure donkey anti human igg h l

4-1BB siRNAs and control siRNA were synthesized by Horizon (Dharmacon Reagents, cat. nos. J-008105-08-0020 and J-008105-09-0020). Activated PBMCs (1 × 10⁶) were transfected with 300 nM individual 4-1BB siRNA or control siRNA using an Amaxa P3 Primary Cell 4D-Nucleofector X Kit S (Lonza, cat. no. V4XP-3032) according to the manufacturer’s protocol. Transfected PBMCs were plated into a 96-well plate for 72 hours. Afterward, 10⁵ transfected PBMCs were preincubated with 2.5 μg of human BD Fc Block (BD Biosciences, cat. no. 564220) for 10 min at RT and then stained with 1 μg of human siglec-4–Fc protein (R&D systems, cat. no. 8940-MG-050) on ice for 30 min followed by AF647 AffiniPure Donkey Anti-Human IgG (H + L) (Jackson ImmunoResearch, cat. no. 709–605-149). 4-1BB was stained using BV421 anti-CD137 (4-1BB) antibody (BioLegend, cat. no. 309819). 4-1BB expression and siglec-4 protein binding were detected using a LSRFortessa flow cytometer (BD Biosciences).

HEK293 cells were transfected with EV, 4-1BBL, or siglec-4 expression plasmids for 48 hours. 4-1BB–HIS protein (0.1 μM) was then incubated with increasing amounts of 4-1BBL–Fc or siglec-4–Fc proteins (0.01 to 1 μM in 100-μl volume of PBS/0.5% BSA buffer) at RT for 30 min. Transfected HEK293 cells were trypsinized and collected, and 5 × 10⁵ cells were incubated with the protein mixtures on ice for 30 min followed by PE-anti-HIS (BioLegend, cat. no. 362603, clone J095G46) staining and FC monitoring to assess the binding of 4-1BB protein to 4-1BBL or siglec-4 on cell surface in the presence of the competitor proteins. To validate that siglec-4 protein binds 4-1BB protein on activated T cells, 0.4 μM siglec-4–Fc protein was incubated with increasing amounts of recombinant soluble 4-1BB–HIS ED protein (0.4 to 10 μM in 100-μl volume of PBS/0.5% BSA buffer) at RT for 30 min. T cells activated by CD3/CD28 treatment were collected, and 5 × 10⁵ cells were incubated with the protein mixtures on ice for 30 min followed by rinsing and detection using AF647 AffiniPure Donkey Anti-Human IgG (H + L) (Jackson ImmunoResearch, cat. no. 709-605-149). FC was used to assess the binding (mean fluorescence intensity) of siglec-4–Fc to activated T cells.