Cd45 magnetic beads isolation kit

Mononuclear cells were isolated as described previously with minor modifications^{9 (link)}. The colon was flushed, cut open longitudinally and incubated in 5 mM EDTA and 10% FBS-containing HBSS buffer to wash out intraepithelial mononuclear cells. The colon was then cut into small pieces, incubated in 5 ml digesting buffer contained 500 U collagenase from Clostridium histolyticum (C2139, Sigma-Aldrich), 2.5 μg DNase I (10104159001, Roche) and 10% FBS (Gibco) at 37 °C for 2 h. The digested colon was homogenized using a 70 μm strainer. CD45⁺ cells were isolated using a CD45 magnetic beads isolation kit (130-052-301, Miltenyi Biotec).

In brief, tissue was cleaned, cut into 1-cm pieces and incubated with 20 ml of pre-warmed PBS containing 1 mM DTT (Thermo Fisher Scientific, P2325), 2.5 mM EDTA and 2.5% fetal bovine serum (FBS) for 20 min at 37 °C. Then, samples were rinsed with PBS and 20 ml of PBS containing 5 mM EDTA and 5% FBS was added for 20 min at 37 °C. Samples were rinsed three times with PBS on a nylon strainer, minced with scissors and digested with 1 mg ml⁻¹ collagenase VIII (Sigma-Aldrich, C2139–5G) and 40 U DNAse (Thermo Fisher Scientific, 90083) in complete RPMI for 40 min at 37 °C. Finally, samples were filtered on 100-μm and 40-μm cell strainers and stained as described above. CD45⁺ cells were isolated using a CD45 magnetic beads isolation kit (130–052-301, Miltenyi Biotec).