Cell suspensions were resuspended in PBE and incubated on ice for 30 min with fluorescently labeled antibodies (Table S2 ) along with 1 mg/ml anti-CD16/32 (24G2; eBioscience).
For detection of cells in early S of the cell cycle, we performed dual nucleotide pulse and staining as previously described (Gitlin et al., 2014 (link)). Briefly, mice were injected i.v. with 1 mg of EdU (A10044; Thermo Fisher Scientific) and 1 h later with 2 mg BrdU (B5002; Sigma). 30 min after the second injection, LNs were harvested, and single-cell suspensions were prepared. After cell surface receptor staining as described above, cells were fixed and permeabilized using BD Cytofix/Cytoperm fixation and permeabilization solution and BD Cytoperm Permeabilization Buffer PLUS, respectively. EdU and BrdU incorporation into DNA was assayed using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen) and FITC BrdU Flow Kit (BD), respectively.
For single-cell sorting, cells were stained as above and index-sorted directly into 96-well plates containing Buffer TCL (Qiagen) supplemented with 1% β-mercaptoethanol using a BD FACS Aria II. Each plate contained all conditions assayed in each replicate. Cells were washed, filtered, and resuspended in PBE before analysis or sorting on BD FACS LSR II, FACS Symphony, or FACS ARIA II cytometers. All data were analyzed using FlowJo software v.10.
For detection of cells in early S of the cell cycle, we performed dual nucleotide pulse and staining as previously described (Gitlin et al., 2014 (link)). Briefly, mice were injected i.v. with 1 mg of EdU (A10044; Thermo Fisher Scientific) and 1 h later with 2 mg BrdU (B5002; Sigma). 30 min after the second injection, LNs were harvested, and single-cell suspensions were prepared. After cell surface receptor staining as described above, cells were fixed and permeabilized using BD Cytofix/Cytoperm fixation and permeabilization solution and BD Cytoperm Permeabilization Buffer PLUS, respectively. EdU and BrdU incorporation into DNA was assayed using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen) and FITC BrdU Flow Kit (BD), respectively.
For single-cell sorting, cells were stained as above and index-sorted directly into 96-well plates containing Buffer TCL (Qiagen) supplemented with 1% β-mercaptoethanol using a BD FACS Aria II. Each plate contained all conditions assayed in each replicate. Cells were washed, filtered, and resuspended in PBE before analysis or sorting on BD FACS LSR II, FACS Symphony, or FACS ARIA II cytometers. All data were analyzed using FlowJo software v.10.
