Blood cell production arises from the activity of hematopoietic stem cells (HSCs), defined by their self-renewal capacity and ability to give rise to all mature blood cell types. The mouse remains one of the most studied species in hematological research, and markers to define and isolate mouse HSCs are well-established. Given the very low frequency of HSCs in the bone marrow, stem cell pre-enrichment by red blood cell lysis and magnetic cell separation is often performed as part of the isolation process to reduce sorting times. Several pre-enrichment strategies are available, differing in their speed, degree of enrichment, final cell yield, and cost. In the current study, we performed a side-by-side comparison and provide a decision tree to help researchers select a pre-enrichment strategy for mouse HSC isolation depending on their downstream application. We then compared different pre-enrichment techniques in combination with metabolomics analysis of HSCs, where speed, yield and temperature during pre-enrichment are crucial factors, and found that the choice of pre-enrichment strategy significantly impacts the number of metabolites detected and levels of individual metabolites in HSCs.
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