Scapin Giorgia, Goulard Marie C, Dharampuriya Priyanka R, Cillis Jennifer L, Shah Dhvanit I
Nationwide Children's Hospital; The Ohio State University College of Medicine; The Ohio State University Comprehensive Cancer Center.
Nationwide Children's Hospital; The Ohio State University College of Medicine; The Ohio State University Comprehensive Cancer Center;
J Vis Exp. 2019 Nov 9(153). doi: 10.3791/60234.
Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a differentiation state to sustain demands of the blood formation. However, it has been difficult to analyze the metabolic status (mitochondrial respiration and glycolysis) of HSPCs due to their limited numbers and lack of optimized protocols for non-adherent, fragile HSPCs. Here, we provide a set of clear, step-by-step instructions to measure metabolic respiration (oxygen consumption rate; OCR) and glycolysis (extracellular acidification rate; ECAR) of murine bone marrow-LineageSca1c-Kit (LSK) HSPCs. This protocol provides a higher amount of LSK HSPCs from murine bone marrow, improves the viability of HSPCs during incubation, facilitates extracellular flux analyses of non-adherent HSPCs, and provides optimized injection protocols (concentration and time) for drugs targeting oxidative phosphorylation and glycolytic pathways. This method enables the prediction of the metabolic status and the health of HSPCs during blood development and diseases.
造血干祖细胞（HSPCs）具有独特的代谢可塑性，这使它们能够从静止状态转变为分化状态，以满足血液生成的需求。然而，由于HSPCs数量有限，且缺乏针对非贴壁、脆弱的HSPCs的优化方案，因此很难分析它们的代谢状态（线粒体呼吸和糖酵解）。在此，我们提供了一套清晰的、分步的操作指南，用于测量小鼠骨髓Lin⁻Sca1⁺c-Kit⁺（LSK）HSPCs的代谢呼吸（氧消耗率；OCR）和糖酵解（细胞外酸化率；ECAR）。该方案可从小鼠骨髓中获得更多数量的LSK HSPCs，提高孵育过程中HSPCs的活力，便于对非贴壁HSPCs进行细胞外通量分析，并为靶向氧化磷酸化和糖酵解途径的药物提供优化的注射方案（浓度和时间）。该方法能够预测血液发育和疾病过程中HSPCs的代谢状态和健康状况。
