Study On Application Of Extracellular Flux Methodology In Profiling Energy Metabolism Of Red Blood Cells

The moment the mature red blood cells(RBCs)leave the bone marrow,it is optimally adapted to perform the binding and transport of oxygen and its delivery to all tissues.Red blood cells modulate oxygen transport,protect hemoglobin from oxidant-induced damage,and maintain the osmotic environment of the cell.Glycolysis is the only energetic metabolic pathway for mature RBCs to obtain ATP which is the energy for RBCs to maintain a number of vital cell functions.Generally,the current methods used to measure RBCs glycolysis are not in living state in real-time.An extracellular flux(XF)methodology has been particularly advantageous,and it is capable of dynamic monitoring of cell energy metabolism in real-time and it has been used in different kinds of cells with nuclear.However,because of the special physiological and structural characteristics of RBCs,they have no nucleus,mitochondria and other organelles and cell shape and size are different from other cells.So the assay conditions were not same as other cells.It may be a challenge for RBCs to assay by the XF methodology and application of the XF technology in erythrocytes and exploration of the assay conditions are necessary.Here,we standardize parameters and describe methods for using XF analysis to measure RBCs glycolysis in isolated,intact,healthy adult human blood.Our research found that a 37? incubation for each blood sample produced a much better glucose response.And it was better to seed the suspended RBCs onto Cell-Tak-coated microplates before the assay,which can improve the glucose response of RBCs.We varied the concentrations of glucose and 2-deoxy-glucose(2-DG)and found that40 mM glucose and 400 mM 2-DG were optimal.We also applied the method to measure the energy metabolism changes in RBCs isolated from storage blood samples.The glucose response and glycolysis capacity of the RBCs decreased to 81.5% and 78.2%,respectively,on Day 42;this result was validated by the ATP content changes.These standardized parameters lay the framework for using XF analysis to profile RBCs glycolysis and the technology will complement information gathered from more extensive and high-throughput metabolomics platforms.