LFA-1/ICAM Blocking in CD4+ T-B Cell Co-culture

CD4⁺ T cells (1 × 10⁵, 100 μL) were co-cultured with autologous B cells (4 × 10⁵, 100 μL) in U-bottom 96 well plates (5 × 10⁵, 200 μL volume/well) as described previously [46 (link)]. Anti-CD11a mAb, 1 μg/mL, or mouse isotype control IgG1 was used to observe the LFA-1/ICAM blocking role when CD4⁺ T cells and B cells were incubating. B cells alone, B cells plus lipopolysaccharide (Sigma, St Louis, MO, USA), and B cells plus lipopolysaccharide and anti-CD11a were cultured as controls. After incubation in RPMI 1640/10% FBS/penicillin/streptomycin at 37°C/5% CO₂ for 8 days, 50 μL fresh media was added to each well. On the fourth day after that, supernatants (200 μL) were harvested and stored at 4°C.