Hematopoietic Regulation in Mice

All the animal experiments were approved by the Bundesministerium für Wissenschaft, Forschung und Wirtschaft (Austrian Federal Ministry of Science and Research, license number GZ BMWFW–66.011/0033–WF/V/3b/2016). Male wild–type (WT) and p27^Y88F/Y88 knock–in (KI) C57BL/6J 7–8–week–old mice were used for the in vivo analysis [20 (link)]. Hematocrit (HCT) and hemoglobin (HGB) values, and red blood cell (RBC) counts of peripheral blood were measured with a scil Vet ABC Hematology Analyzer. Reticulocyte analysis was performed as described by Lee et al. [25 (link)]. Briefly, 1 μL of peripheral blood was stained with Thiazole orange for an hour at room temperature and cells were analyzed on a BD LSR flow cytometer. Circulating plasma Epo levels from 6 male WT and 5 male knock–in mice were measured using ELISA according to the manufacturer’s protocol (Mouse Erythropoietin Quantikine ELISA, R&D SYSTEMS, Minneapolis, MN, USA). For the RBC survival analysis, mice were injected intravenously with 1 mg NHS–biotin (Sigma–Aldrich, St. Louis, MO, USA). An amount of 5 μL of blood was withdrawn from the tail after one hour (set as the 0 time point) and at the indicated time points for the following 7 weeks. The cells were stained with Ter119–PE and streptavidin–APC and analyzed on a BD LSR flow cytometer.