Rat anti mouse cd31

Pregnant female mice received intraperitoneal (i.p.) injections with 50μg of 5-ethynyl-2′-deoxyuridine (EdU)/g body weight (Thermo Fisher Scientific – A10044), 16 h prior to tissue collection. Lz dissociation into single cells was performed as above. Cells re-suspended at a concentration of 1000 cells/μl were incubated for 30 min at 4°C with 1 μl Red LIVE/DEAD Fixable Dead Cell Stain (Thermo Fisher Scientific – L23102). After one wash in PBS, the cells were pre-incubated for 20 min at 4°C in the dark with unlabelled rat anti-mouse CD16/32 (BioLegend – 101320, 1 μg/million cells), then for 1 h at 4°C in the dark with a 1:1 mix of rat anti-mouse CD41 (labelled with BV421) (BioLegend – 133911; 0.25 μg per million cells) and rat anti-mouse CD31 (labelled with AF647) (BioLegend – 102516; 0.25 μg per million cells) in staining buffer. After two washes with staining buffer, the cells were stained using the Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Thermo Fisher Scientific – C10420), according to manufacturer’s instructions. Flow cytometry analysis was performed using a BD LSRFortessa cell analyser (BD Biosciences). FSC files were analysed with the FlowJo_V10 software, using single-cell discrimination and gating based on single-stained controls. Proliferating FPEC were identified as viable EdU⁺/CD31⁺/CD41^- cells.