Isolation and Purification of Colonic Macrophages

Macrophages were isolated from colonic tissues according to a published protocol [57 (link)]. Briefly, the colon tissues were minced into a 1.5-cm pieces and washed with warm Ca²⁺ and Mg²⁺-free PBS. Afterward, the samples were treated with pre-warmed 2 mM EDTA in Ca²⁺ and Mg²⁺-free HBSS (Thermo Fisher Scientific, 88,284) with 2% FBS for 20 min at 37°C to remove the epithelial layer. For digestion of intestinal tissue, the samples were digested in pre-warm collagenase VIII (Sigma‐Aldrich, C2139), DNAse I (Sigma‐Aldrich, 4,536,282,001) solution for 20 min at 37°C and then centrifuged at 250 × g for 5 min. For antibody staining, cell pellets were incubated in the flow cytometry staining buffer and incubated with 2.5 ug purified anti-mouse F4/80 antibody (Biolegend, 157,310) and anti-mouse PTPRC/CD45 (Biolegend, 103,155) for 30 min on ice. For storing, 3 × 10⁵ cells were isolated and washed once with PBS and then sorted by a BD FACSAria Fusion Flow cytometer. All data and figures were analyzed and created using the FlowJo Software.