Edta coated capillary tubes

For proliferation assays, BrdU (Sigma) was administered at 0.5 mg ml⁻¹ in drinking water. For HSC exhaustion experiments, 5-FU (Sigma) was injected at 150 mg per kg body weight intraperitoneally (i.p.) once or every 10 days. For myelo-suppressive treatments, 5-FU was injected once (150 mg kg⁻¹ i.p.). To assess haematological recovery, peripheral blood (50 μl) was collected into EDTA-coated capillary tubes (Sarstedt), and differential counts were measured using a Pentra 80 hematologic analyser (Horiba). To assess HSPC quiescence, hydroxyurea (Sigma) was injected twice (8 h apart) at 900 mg per kg body weight i.p. as described earlier4 (link). For in vivo inhibition of TGFβ, a neutralizing antibody (clone 1D11) or a non-targeted control antibody (clone HRPN; both IgG1 from BioXcell) were intravenously injected at 100 μg per mouse once every 2 days for a total of 2 weeks. To block E-selectin, mice were treated with the same doses as above of an anti-E-selectin blocking antibody (clone 9A9; BioXcell) following the same schedule. To deplete macrophages, mice were intravenously injected with 100 μl of liposomes loaded with clodronate 8 days before analysis (Clodronateliposomes.com).

In a separate group of animals, we determined blood alcohol levels (BALs) after an IP injection of 0.6 and 1.2 g/kg alcohol. Thirty minutes after injection, blood samples were collected from the lateral tail vein in EDTA-coated capillary tubes (Sarstedt, Numbrecht, Germany) and immediately stored on ice. In addition, to explore the metabolism of alcohol over time, animals were treated with 0.6 g/kg for blood sampling at 5, 10, 15, 30, 60, and 120 min after injection. Blood samples were spun at 3000 rpm for 20 min (at 4 °C) and plasma was stored at −20 °C until blood alcohol analysis. BALs (mg/dl) were determined using an NAD-ADH reagent kit (Sigma-Aldrich, Schnelldorf, Germany) and a standard curve for quantitation.