【摘要】 目的:探索Seahorse XFe细胞能量代谢检测仪检测红细胞能量代谢的实验条件及方法,对传统红细胞能量代谢检测方法进行补充。方法:向Seahorse细胞培养板添加不同数量的红细胞,并根据倒置显微镜下红细胞分布状态确定最优细胞检测密度;利用Seahorse XFe24细胞能量代谢检测仪,在37℃条件下,向活体红细胞依次添加不同浓度的葡萄糖(Glucose)以及2-脱氧-D-葡萄糖(2-Deoxy-D-Glucose,2-DG)并连续监测其胞外酸化率(Extracellular acidification rate,ECAR)的变化,计算其基础糖酵解(Baseline glycolysis)、葡萄糖反应性(Glucose response)以及糖酵解能力(Glycolytic capacity)3个指标。结果:通过镜下观察红细胞分布状态得到最佳的红细胞检测密度为(5～6)×10~8/well,通过对基础糖酵解、葡萄糖反应性以及糖酵解能力3个指标的分析得到最佳葡萄糖和2-DG作用终浓度分别为10 mM和100 mM。结论:应用Seahorse XFe细胞能量代谢检测仪能够保证检测过程中细胞的完整性及活性,检测结果具有连续性及动态性,为红细胞能量代谢的相关研究提供了一个更为全面的研究体系。更多还原

【Abstract】 Objective:To explore the experimental conditions and methods of detecting erythrocyte energy metabolism with Seahorse XFe cell energy metabolism detector and supplement the traditional methods of detecting erythrocyte energy metabolism. Methods:Different numbers of red blood cells were added to the Seahorse cell culture plate, and the optimal cell density was determined according to the distribution of red blood cells under the inverted microscope. Under the condition of 37 ℃, adding glucose and 2-deoxidation-D-Glucose(2-DG) of different concentrations to living red blood cells in order and their extracellular acidification rates( ECAR) were continuously monitored, with the indexes of baseline glycolysis, glucose response and glycolytic capacity calculated. Results:The optimal red blood cell detection density was(5-6) X10~8/well by observing the distribution of red blood cells under the microscope and the final concentration of Glucose/2-DG was 10 mM/100 mM by analyzing the three indicators of baseline glycolysis, glucose response and glycolytic capacity. Conclusion:The application of Seahorse XFe cell energy metabolism detector can ensure the integrity and activity of cells in the detection process, and the test results are continuous and dynamic, which is an effective supplement to the traditional detection methods. It provides a more comprehensive research system for the related research of red blood cell energy metabolism.更多还原