Tail bleeding time

A 3 mm tail-tip transection method was used to assess haemostasis in 20–25 g male and female anaesthetized and ventilated mice²². Following transection, the mouse tail was immediately immersed into warmed (37 °C) saline. The bleeding time was determined as the time from the tail transection to the moment the blood flow stopped for more than 120 s. A bleeding time beyond 30 min was considered as the cutoff time for the purpose of statistical analysis. Red blood cells were pelleted and lysed in 1 ml H₂0. Haemoglobin was quantified by absorbance at 575 nm (Beckman DU530 Life Science UV/Vis Spectrophotometer). Following cessation of bleeding, the incision was monitored for a further 120 s, and further bleeding within this period classified as ‘re-bleeding’. The duration of rebleeding was monitored as described above. Non-genotyped mice were used for the 3 mm tail-tip assay, and were subsequently genotyped using tail tissue collected post euthanasia.