Evaluation of glycolytic rates in human hematopoietic stem/progenitor cells after target gene depletion

STAR Protoc. 2021 Jun 11;2(2):100603. doi: 10.1016/j.xpro.2021.100603. eCollection 2021 Jun 18.

Abstract

This protocol describes how to evaluate cellular metabolic state, including the glycolytic activity, in live CD34+ hematopoietic stem/progenitor cells (HSPCs) upon depletion of a specific target gene. We detail the procedure of enriching mononuclear cells from human umbilical cord blood samples, isolation of CD34+ HSPCs using positive immunomagnetic separation, and the analysis of glycolytic activity in lentivirally transduced CD34+ HSPCs using Seahorse Assay. This protocol can be applied to evaluate potential aerobic glycolysis gene targets for cancer therapy. For complete details on the use and execution of this protocol, please refer to Qing et al. (2021).

Keywords: Cancer; Cell isolation; Cell-based Assays; Gene Expression; Metabolism; Molecular Biology; Stem Cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, CD34 / immunology
  • Glycolysis
  • HEK293 Cells
  • Hematopoietic Stem Cells / cytology
  • Hematopoietic Stem Cells / immunology
  • Hematopoietic Stem Cells / metabolism*
  • Humans
  • Immunomagnetic Separation
  • Transduction, Genetic

Substances

  • Antigens, CD34