Optimised Workflows for Profiling the Metabolic Fluxes in Suspension vs. Adherent Cancer Cells via Seahorse Technology

Oxidative phosphorylation and glycolysis are the main ATP-generating pathways in cell metabolism. The balance between these two pathways is frequently altered to carry out cell-specific activities in response to stimuli involving activation, proliferation, or differentiation. Despite being a useful tool for researching metabolic profiles in real time in relatively small numbers of cancer cells, the main Agilent Seahorse XF Pro Analyzer (Agilent Technologies, Santa Clara, CA, USA) guideline is currently not fully detailed in the distinction between suspensions vs. adherent cancer cells. This article provides step-by-step protocols for profiling metabolic fluxes in suspension vs. adherent cancer cells via Seahorse technology, including adjustments for normalisation of data on the basis of the number of viable cells or the total protein content. Owing to the adaptations of plates, reagents, cell count, and protein quantification, it is possible to (i) analyse both adherent and suspension cells with a single instrument; (ii) conduct all experiments in 96-well plates, thus using fewer cells, media, and reagents; (iii) determine the effect of a drug or compound directly on cell metabolism; (iv) normalise data on the basis of the number of viable cells or the total protein content via a spectrophotometer; and (v) achieve notable savings in cost and time.