Citrate solution

Tartrate resistant acid phosphatase (TRAP) staining was performed using a commercially available acid phosphatase kit from Sigma-Aldrich (Cat No. 387, Sigma Aldrich, Schnelldorf, Germany). The staining was performed according to the manufacturer's instructions. In detail, after aspiration of the culture medium, the cells were fixed with 100 μL fixative solution (3.316 mL acetone, 1.276 mL citrate solution (Sigma Aldrich, Schnelldorf, Germany), and 0.408 mL 37% formaldehyde) for 30 s. Thereafter the samples were washed twice with 200 μL PBS. Then, 100 μL of a freshly prepared diazotized Fast Garnet GBC Base Naphthol AS-BI phosphoric acid staining solution (Sigma Aldrich, Schnelldorf, Germany) was added to the wells followed by incubation at 37 °C and 5% CO₂. After 60 min the staining solution was aspirated, the surfaces were washed twice with 200 μL dH₂O and dried overnight at RT. The transmitted light microscope VisiScope IT 404 (VWR International, Leuven, Belgium) and the software ZEN 2.3 (blue edition) from Carl Zeiss Microscopy GmbH (Zeiss, Oberkochen, Germany) were used to analyze the PS reference. The scaffolds were analyzed with the SteREO Discovery.V20 stereomicroscope and the ZEN 2012 (blue edition) software (both Zeiss GmbH, Oberkochen, Germany).

For coagulation analyses, freshly collected blood was incubated with 1/10 v/v citrate solution (Sigma-Aldrich; Hamburg, Germany) to inhibit blood coagulation. Afterwards, the blood was centrifuged for 20 minutes at 600 xg, the obtained plasma was diluted ¼ with PBS, and used for blood coagulation assays, using a KC4 Delta Coagulometer (Tcoag; Bray, Ireland).
To determine the prothrombin time (PT), 50 μL plasma was incubated for 1 minute at 37°C. After adding 100 μL thromborel S (Siemens Healthcare; Marburg, Germany) the time until complete clotting of the sample occurred was measured.
For analyzing the partial thromboplastin time (aPTT), 50 μL plasma was incubated with 50 μL pathromtin SL (Siemens Healthcare; Marburg, Germany) for 5 minutes at 37°C. The reaction was started after adding 50 μL of a 0.025 M calcium chloride solution (Siemens Healthcare; Marburg, Germany). The time until complete clotting of the sample occurred was measured.

After injecting a lethal dose of pentobarbital intraperitoneally (i.p.), intracardial blood was obtained for later analysis on the Sysmex XP Hematology Analyzer or HEMAVET using a syringe coated with citrate solution (Sigma) and a 26 G needle.
For smaller amounts of blood at multiple points in time, e.g., for analyzing erythrocyte turnover and stress erythropoiesis, mice were gently restrained, while blood was drawn by scratching the Vena caudalis mediana and immediately transferred to an EDTA-coated reaction vessel.