Pe conjugated annexin 5

PtdSer exposure was detected using Alexa Fluor-647, FITC or PE conjugated Annexin-V (all 1:100, BioLegend, UK) diluted in AnnV binding buffer (Hanks Balanced Salt Solution H6648 with 2.5mL 1M CaCl₂) and incubated for 15-minutes. PI was used as above. Cells were designated non-apoptotic (AnnV^-PI^-), early (AnnV⁺PI^-) or late (AnnV⁺PI⁺) apoptotic. Staining was assessed by flow cytometry on a BD FACSCalibur (10,000 events collected) and analysed using FlowJo (TreeStar).

To isolate cells of different stages of differentiation directly from primary fetal livers at E12.5–E13.5, 5–15 fetal livers were pooled together, mechanically dissociated in staining buffer (PBS, 0.2% BSA, 5 mM glucose) and strained through a 30-μM filter. Cells were stained at 4 °C with rabbit IgG (200 μg/ml, Jackson Laboratories) to block Fc receptors. Cells were then incubated with PE-Cy7 conjugated anti-CD71 (RI7217 BioLegend, 1:2000), APC conjugated anti-Ter119 (RUO, BD Biosciences; 1:200), BB700 conjugated CD117 (104D2, BD Biosciences, 1:100), PE-conjugated Annexin V (BioLegend, 1:100), a panel of 5 FITC-conjugated lineage antibodies (anti-CD41, anti-CD45R, anti-CD3e, anti-CD11b, and anti-Ly-6G/6C, all at 1 μg/ml, all BD Biosciences), Cells were then resuspended in FACS running buffer (staining buffer with the addition of 2  mM EDTA). 0.66  μg/ml Hoechst (Invitrogen) was added prior to sorting, which was performed on a BD FACSAria Fusion machine with a 100 μm nozzle. Annexin V was only added for the experiments on the Lrf^−/− mice. Single color controls and FMOs were used for calibration.