Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their energetic needs. However, under stress conditions such as infection or blood loss, HSCs become proliferative and rapidly produce downstream progenitor cells, which in turn further differentiate, ultimately producing mature blood cells. During this transition and differentiation process, HSCs exit from quiescence and rapidly undergo a metabolic switch from glycolysis to oxidative phosphorylation (OxPHOS). Various stress conditions, such as aging, cancer, diabetes, and obesity, can negatively impact mitochondrial function and thus can alter the metabolic reprogramming and differentiation of HSCs and progenitors during hematopoiesis. Valuable insights into glycolytic and mitochondrial functions of HSCs and progenitors under normal and stress conditions can be gained through the assessment of their extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which are indicators of cellular glycolysis and mitochondrial respiration, respectively. Here, a detailed protocol is provided to measure ECAR and OCR in mouse bone marrow-derived lineage-negative cell populations, which include both hematopoietic stem and primitive progenitor cells (HSPCs), using the extracellular flux analyzer. This protocol describes approaches to isolate lineage-negative cells from mouse bone marrow, explains optimization of cell seeding density and concentrations of 2-deoxy-D-glucose (2-DG, a glucose analog that inhibits glycolysis) and various OxPHOS-targeted drugs (oligomycin, FCCP, rotenone, and antimycin A) used in these assays, and describes drug treatment strategies. Key parameters of glycolytic flux, such as glycolysis, glycolytic capacity, and glycolytic reserve, and OxPHOS parameters, such as basal respiration, maximal respiration, proton leak, ATP production, spare respiratory capacity, and coupling efficiency, can be measured in these assays. This protocol allows ECAR and OCR measurements on non-adherent HSPCs and can be generalized to optimize analysis conditions for any type of suspension cells.