Microcapillary tubes

WT and KO mice obtained from SLC48A1 HET crosses were weaned at 21 days of age (P21) and placed on their respective diets, supplemented with deionized water. Standard rodent diet was obtained from Envigo and the two ppm (TD.09127) Fe diet was custom ordered from Envigo, Madison, WI. Body weights and feed intake per cage were measured weekly to ensure the conditions of the animals. Blood was collected by retro-orbital bleeding using microcapillary tubes (Fisher Scientific, cat. number 22–362566). At the end of five weeks, mice were sacrificed by cardiac perfusion using Dulbecco's phosphate-buffered saline (DPBS) (Gibco, cat. number 14190250) under anesthesia (10% ketamine, 8% xylazine mix). Prior to perfusion, whole blood was collected into tubes and allowed to clot at room temperature for 45 min and serum was separated from the sample by centrifugation at 2000 g for 10 min. For the prolonged dietary iron study where the Kaplan Meier survival curve was obtained, whole blood hematocrits were obtained every two weeks until week 14. All surviving KO mice were sacrificed when the survival rate fell below 50% (~week 16), while WT mice were kept on the diets until week 18.

Mice were anesthetized using a vaporized 4% isofluorane/96% oxygen mixture and 100 μL blood collected by retro-orbital bleed with heparinized micro-capillary tubes (Fisher Scientific, Hampton, NH, USA). Samples were transferred immediately to polypropylene micro-centrifuge tubes. Blood samples were incubated for 30 min at room temperature then allowed to clot overnight at 4 °C, before centrifugation at 13,000 g for 15 min at 4 °C. Sera were withdrawn with a pipette and stored in aliquots at −20 °C.

WT and KO mice obtained from SLC48A1 HET crosses were weaned at 21 days of age (P21) and placed on their respective diets, supplemented with deionized water. Standard rodent diet was obtained from Envigo and the two ppm (TD.09127) Fe diet was custom ordered from Envigo, Madison, WI. Body weights and feed intake per cage were measured weekly to ensure the conditions of the animals. Blood was collected by retro-orbital bleeding using microcapillary tubes (Fisher Scientific, cat. number 22–362566). At the end of five weeks, mice were sacrificed by cardiac perfusion using Dulbecco's phosphate-buffered saline (DPBS) (Gibco, cat. number 14190250) under anesthesia (10% ketamine, 8% xylazine mix). Prior to perfusion, whole blood was collected into tubes and allowed to clot at room temperature for 45 min and serum was separated from the sample by centrifugation at 2000 g for 10 min. For the prolonged dietary iron study where the Kaplan Meier survival curve was obtained, whole blood hematocrits were obtained every two weeks until week 14. All surviving KO mice were sacrificed when the survival rate fell below 50% (~week 16), while WT mice were kept on the diets until week 18.