Easy step human biotin isolation kit

Human fetal spleen cells were isolated by a 30-min digestion with freshly prepared medium in 1 mg mL⁻¹ collagenase IV (Gibco) and 10 mg mL⁻¹ DNAse (Roche) in complete RPMI [RPMI medium (GIBCO) without antibiotics, 10% FBS (GIBCO), 1 mM sodium pyruvate (Life Technologies), 2 mM l-glutamine (Life Technologies), 1× nonessential amino acids (Life Technologies) and 10 mM HEPES (Life Technologies)].
Digested splenic cells were gently pressed through a 70-μm strainer, and cells were separated in a 20%:40%:80% Percoll density gradient at 400g for 40 min; APCs were recovered at the 20–40% interface. All cells were washed twice with cRPMI medium. Viability was measured with Aqua dye (Invitrogen) using flow cytometry. APCs from human fetal spleen were enriched by positive selection using Easy Step Human Biotin Isolation kit (STEMCELL Technologies) and the biotinylated human HLA-DR monoclonal antibody. Cells were seeded into 96-well plates and incubated with a multiplicity of infection (MOI) of 10 bacterial cells in antibiotic-free cRPMI for 4 h at 37°C with 5% CO₂ and 4% O₂ to mimic hypoxic conditions in the fetal intestine⁵⁸ (link) and normalize for bacterial growth. Cells were harvested by fixation and stained for flow cytometry as described below.