In vivo EdU incorporation

For analysis of EdU incorporation in GFP+ cells, mice on a 3-day course of 3 mg ml⁻¹ Dox in drinking water received a single intraperitoneal injection of 0.5 mg EdU in PBS per 10 g mouse weight. After 24 h, mice were killed, and mammary epithelial cells were dissociated from freshly harvested tissue. Single cells were fixed with 4% paraformaldehyde (PFA), stained using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry assay kit (ThermoFisher Scientific) and analysed on a BD LSRFortessa. For experiments comparing EdU incorporation in mammary epithelial cells versus small intestine epithelial cells, mice received a single intraperitoneal injection of 0.5 mg EdU in PBS per 10 g mouse weight, and were killed after 24 h and analysed as above. For experiments comparing EdU incorporation in mammary epithelial cells from Brca2 mutant mice versus heterozygous controls, mice received a single intraperitoneal injection of 0.3 mg EdU in PBS per 10 g mouse weight. After 3 h, mice were killed and mammary epithelial cells were dissociated from freshly harvested tissue, fixed with 4% PFA, stained using the Click-iT EdU Alexa Fluor 488 Flow Cytometry assay kit (ThermoFisher Scientific) and analysed on a BD FACScan.