Isolation and Enrichment of Antigen-Presenting Cells

Thymi were isolated and digested with 125 mg/ml collagenase D and 62.5 µg/ml DNaseI for 20 min with gentle rocking at 37°C. Digests were filtered through a 70-µm filter cap strainer (BD Falcon), resuspended in 1 ml high-density (1.115 g/ml) Percoll solution (GE healthcare), and layered with 1 ml low-density (1.065 g/ml) Percoll solution followed by 1 ml PBS. This Percoll gradient was centrifuged at 2,700 rpm for 30 min at 4°C to enrich antigen-presenting cells. Cells between the PBS and low-density Percoll layer were isolated and used for flow cytometric analysis as described in our previous study (Oh et al., 2013 (link)) or enriched further by using magnetic beads conjugated with anti-CD11c antibody (Miltenyi Biotec).