Pe anti mouse f4 80

Tumor tissues were resected from mice and minced into small pieces and then were lysed by 1 mg/ml collagenase IV (Sigma, United States) and DNase I (Invitrogen, United States) for 1 h at 37°C. Afterward, the tissue medium was filtrated using a 70-μm filter screen to obtain single-cell suspensions. The cell suspensions were stained with antibodies for 30 min and washed three times by PBS and then were subjected to FCM analysis. The following reagents and antibodies were used in FCM analysis.
Panel A: LIVE/DEAD™ Fixable Stain (Invitrogen, California, United States), anti-mouse CD45-BV605 (Biolegend, California, United States), anti-mouse CD3-PE-cy7 (Biolegend, California, United States), anti-mouse CD4-Efluor450 (BD, New Jersey, United States), anti-mouse CD8-Percp-cy5.5 (Biolegend, California, United States), anti-mouse CD19-BV650 (Biolegend, California, United States), and anti-mouse NK1.1-PE (Biolegend, California, United States).
Panel B: LIVE/DEAD™ Fixable Stain (Invitrogen, California, United States), anti-mouse CD45-BV605 (Biolegend, California, United States), anti-mouse CD11b-Percp-cy5.5 (Biolegend, California, United States), anti-mouse F4/80-PE (Biolegend, California, United States), and anti-mouse Ly6G-APC (Biolegend, California, United States).

Macrophage efferocytosis assays were performed as previously described [24 (link)–26 (link)]. Briefly, apoptotic epithelial cells (with an apoptotic rate greater than 70%) were prepared (Additional file 1: Fig. S1) and labelled with CFSE (5,6-carboxyfluorescein diacetate, succinimidyl ester, Cell Division Tracker Kit, BioLegend, USA). Apoptotic A549 cells were cocultured with primary human lung macrophages or THP-1 cells, while apoptotic MLE12 cells were cocultured with primary mouse lung macrophages. After coculturing with sufficient CFSE-labelled apoptotic epithelial cells (apoptotic cells: Mø > 10:1), macrophages were labelled with anti-human CD68-APC (1:40 dilution for flow cytometry and 1:25 dilution for immunofluorescence, BioLegend) or anti-mouse F4/80-PE (1:50 dilution for flow cytometry and 1:25 dilution for immunofluorescence, BioLegend). The ratio and capacity of macrophage efferocytosis were analysed by fluorescence microscopy (Leica DM6 FS, Germany) and flow cytometry (FACS Verse, BD), respectively, and determined by detecting the CFSE-positive ratio and mean fluorescence intensity (MFI) of CFSE in macrophages, respectively.

Small intestinal lamina propria (SILP) cells were isolated using a Lamina Propria
Dissociation Kit (Miltenyi Biotec) and analyzed using a BD FACS CANTO II. The
following primary Abs were used: anti-mouse CD45 A647 (Biolegend 30-F11),
anti-mouse CD11b PE-Cy7 (Biolegend), anti-mouse Ly-6G PerCP-Cy5.5 (Biolegend),
anti-mouse F4/80 PE (Biolegend).