Treated or untreated young mice (8–12 weeks of age) were injected intraperitoneally with 100 μl of colchicine (0.5% w/v in water, Sigma). After 30 min the mice were culled by cervical dislocation, femurs were harvested and placed in ice-cold PBS. Bone marrow cells were flushed with 10 ml of pre-warmed hypotonic solution (75 mM KCl, 37 °C) through a 70-μm cell strainer and incubated for 15 min in a water bath at 37 °C. After the incubation, 1 ml of fixative (3:1 methanol:acetic acid) was added dropwise to the hypotonic buffer, and mixed by gentle inversion of the tube. The tubes were spun down for 10 min at 250g and the supernatant was aspirated, leaving 50 μl and the cell pellet in the tube. The cells were resuspended by flicking the base of the tube very gently, 3 ml of fixative were added dropwise and the volume was made up to 10 ml by pipetting fixative down the side of the tube. The cells were incubated at room temperature for 30 min and stored at −20 °C until further use.
