Fcs express 6 and 7

Immunophenotypic analysis was performed using one of the following instruments: BD FACSCanto II, BD LSRFortessa II, BD FACSymphony A5, Beckman Coulter Cytoflex S or Beckman Coulter Cytoflex LX. Antibodies used for immunophenotypic analysis are listed in Supplementary Data 7. Cells were incubated with a human (Miltenyi Biotec, #130-059-901, dilution 2:100) and mouse FcR blocking reagent (BD #553141, dilution 1:100) prior to staining with antibody cocktail. All steps were performed at 4 °C in PBS 2% FBS. Data were analyzed with FCS Express 6 and 7 (DeNovo Software). Staining for cell cycle analysis was performed by fixing and permeabilizing cells with the eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set as per manufacturer’s indications, followed by 30 min incubation at +4^oC with human FcR blocking reagent (Miltenyi Biotec, #130-059-901, dilution 2:100) and overnight staining at +4^oC with Ki67-AF647 (BD #558615, dilution 2:100). Samples were acquired on the FACSymphony A5 with low flow rates after addition of Hoechst 33342. Staining for EdU incorporation was performed with the Click-iT™ Plus EdU Pacific Blue™ Flow Cytometry Assay Kit (TermoFisher Scientific #C10636) following the manufacturer’s indications.

The flow cytometry data were analyzed with FCS Express 6 and 7 (De Novo Software, Pasadena, CA, USA). The results in this study are presented as mean ± standard deviation (SD). The statistical analyses were performed in Prism 8 (GraphPad Software, San Diego, CA, USA) using an unpaired two-tailed T test. A difference with a P value less than 0.05 was considered statistically significant.