Mouse erythropoietin quantikine elisa

Sera (20–60 μL) were prepared from the peripheral blood collected from 12–14 M-old mice by cheek bleeding and stored at -80°C. The samples were diluted six-fold, and EPO measurement was done using Quantikine ELISA Mouse Erythropoietin (R&D Systems, Minneapolis, MN), according to manufacturer’s instructions. All samples were measured in duplicate.

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 An amount of 5 μL of blood was withdrawn from the tail after one hour (set as the 0 time point) and at the indicated time points for the following 7 weeks.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.