Anti lineage cocktail

Anti-Nono (Cat# 11058-1-AP and 66361-1-Ig) was from Proteintech. Anti-Elk3 (Cat# NBP1-83960) was from Novus Biologicals. Anti-Tmem241 (Cat# 203644-T32) was from Sinobiological. Anti-Lineage cocktail (Cat# 88-7772-72), Anti-CD127 (A7R34), anti-Sca-1 (D7), anti-Flt3 (A2F10), anti-α₄β₇ (DATK32), anti-Id2 (ILCID2), anti-PLZF (Mags.21F7), anti-Eomes (Dan11mag), anti-NKp46 (29A1.4), anti-NK1.1 (PK136), anti-CD45 (30-F11), anti-CD25 (PC61.5), anti-Gata3 (TWAJ), anti-RORγt (AFKJS-9), anti-Bcl11b (1F8H9), anti-CD3 (17A2), anti-CD19 (1D3), anti-KLRG1 (2F1), anti-CD90 (HIS51), anti-IL-22 (IL22JOP), anti-Ki67 (SolA15), anti-CD45.2 (104), anti-BrdU (BU20A), anti-PD-1 (J43) and anti-CD45.1 (A20) were purchased from eBiosciences (San Diego, USA). Anti-c-Kit (2B8), anti-CD150 (TC15-12F12.2), anti-CD48 (HM48-1), and anti-CD49a (HMα1) were purchased from Biolegend (California, USA). Anti-β-actin (Cat# RM2001) was purchased from Beijing Ray Antibody Biotech. Paraformaldehyde (PFA) and 4’,6-diamidino-2-phenylindole (DAPI) were from Sigma. IL-22 ELISA kit was purchased from Neobioscience.

Peritoneal cells were extracted by lavage. Splenic cells were prepared by straining through a 70-µm mesh, and then red blood cells were lysed with red cell lysis buffer (Biolegend). For flow cytometry, dead cells were excluded using fixable viability dye 780 (eBioscience), and Fc receptors were blocked with anti-CD16/32 antibody (93; Biolegend). Fluorochrome-conjugated anti-CD19 (6D5), anti-CD5 (53-7.3), anti-CD23 (B3B4), anti-IgM (RMM-1), anti-B220 (RA3-6B2), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD93 (AA4.1; eBioscience), antilineage cocktail (eBioscience), anti-CD21 (7E9), and anti-CD36 (HM36) antibodies were used for surface staining (all from Biolegend unless otherwise specified). Flow cytometry was performed on LSR II or Fortessa X20 instruments (BD Biosciences). Flow sorting was performed on a flow cytometer (FACSAria III; BD Biosciences). Intracellular staining for anti–active caspase-3 (C92-605; BD Biosciences) and Ki67 (11F6; Biolegend) was performed after fixation and permeabilization (FoxP3/transcription factor buffer set; eBioscience).