Protocol for separating cancer cell subpopulations by metabolic activity using flow cytometry

Cells, even from the same line, can maintain heterogeneity in metabolic activity. Here, we present a protocol, adapted for fluorescence-activated cell sorting (FACS), that separates resuspended cells according to their metabolic rate. We describe steps for driving lactate efflux, which produces an alkaline transient proportional to fermentative rate. This pH signature, measured using pH-sensitive dyes, identifies cells with the highest metabolic rate. We then describe a fluorimetric assay of oxygen consumption and acid production to confirm the metabolic contrast between subpopulations. For complete details on the use and execution of this protocol, please refer to Blaszczak et al.¹.