Thymus Tissue Dissociation and Flow Cytometry

Thymus tissue was incubated for 1 h at 37°C in collagenase digestion mixture (0.1 mg/ml collagenase type 4 in calcium-replete HBSS containing 10% FCS and 200 U/ml DNase). After homogenization and filtration, the resultant cells were stained and analyzed using an LSRII flow cytometer together with FlowJo software (Tree Star) as has been described [35 (link),36 (link)]. Biotin-conjugated anti-CD90.2 (53–2.2), eFluor605NC-conjugated anti-CD19 (1D3), APC-conjugated anti-CD4 (GK1.5), eFluor450-conjugated anti-CD8 (53–6.7), and PE-Cy7-conjugated anti-CD3 (145–2C11) were purchased from eBioscience, whereas APC-Cy7-conjugated anti-CD45 (30-F11) was obtained from BD Pharmingen. Biotin-conjugated antibodies were detected using streptavidin conjugated with PE-TexasRed (Invitrogen). Mann–Whitney t-tests (with a 95% confidence interval) were performed using Prism 6.0 (GraphPad Software, La Jolla, USA). All P-values are two-tailed (*P < 0.05, **P < 0.01, and ***P < 0.001).