The micronucleus assay was performed essentially as described previously38. Treated or untreated mice (8–12 weeks of age) were bled and 62 μl blood was mixed with 338 μl PBS supplemented with 1,000 U ml−1 of heparin (Calbiochem). 360 μl of blood suspension was then added to 3.6 ml of methanol at −80°C and stored at −80°C for at least 12 h. 1 ml of fixed blood cells was then washed with 6 ml of bicarbonate buffer (0.9% NaCl, 5.3 mM NaHCO3). The cells were resuspended in 150 μl of bicarbonate buffer and 20 μl of this suspension was used for subsequent staining. 72 μl of bicarbonate buffer, 1 μl of FITC-conjugated CD71 antibody (GenTex, clone R17217.1.4) and 7 μl RNase A (Sigma) were premixed and added to 20 μl of each cell suspension. The cells were stained at 4 °C for 45 min, followed by addition of 1 ml bicarbonate buffer and centrifugation. Finally, cell pellets were resuspended in 500 μl bicarbonate buffer supplemented with 5 μg ml−1 propidium iodide (Sigma). The samples were analysed immediately on an LSRII FACS analyser (BD) and the data analysed with FlowJo v10.0.7.
