Flow cytometry

Cells were trypsinized, washed in 2% BSA FACS buffer and stained in primary antibody or directly conjugated antibodies at 5 μg/mL at 4°C for 45 minutes as specified in the Antibodies list. Cells were washed twice with 0.5% BSA FACS buffer and incubated in secondary antibody (1:250 Anti-mouse AlexaFluor 568, A10037, RRID:AB_2534013, Life Technologies) in 0.5% BSA FACS buffer with fixable viability dye (FVD450nm; 65-0863-14, eBioscience) at 1:250. After 30 minutes at 4°C and three washing steps with 2% BSA FACS buffer, cells were fixed with 2% formalin (HT501128, Sigma-Aldrich) in 2% BSA FACS buffer for 10 minutes and washed. Mouse omenta were digested in HBSS (9374543, Gibco) supplemented with collagenase (C9263, Sigma) and DNAase I for 20 minutes at 37°C and filtered through a 70 μm strainer. Cells were washed and resuspended in FACS buffer (PBS + 2% FBS + 2 mmol/L EDTA). The cell suspension was stained in FACS buffer for 30 minutes at 4°C with the antibodies listed in Supplementary Table S1. Cells were washed and stained with FVD506 (65-0866-14, eBioscience) for 25 minutes at 4°C. After fixation, cells were analyzed on a LSR Fortessa II (BD Biosciences) and results analysed with FlowJo v10.2, RRID:SCR_008520, (Treestar Inc.).