Sorting cells alters their redox state and cellular metabolome

Area of Science:

• Cellular biology
• Metabolomics
• Biochemistry

Background:

• Cellular metabolomics is crucial for understanding biological processes.
• Fluorescence-activated cell sorting (FACS) is a common method for purifying cells.
• Concerns exist regarding metabolic artifacts introduced by cell culture and purification.

Purpose of the Study:

• To investigate the impact of FACS on cellular metabolism and oxidative stress.
• To determine if FACS-purified cells accurately represent their pre-sorted metabolic state.

Main Methods:

• Astrocytes were subjected to FACS.
• Metabolomic analysis was performed using liquid chromatography/mass spectrometry.
• Levels of glutathione redox couple (GSSG/GSH), NAD(P)H/NAD(P)+, and reactive oxygen species were measured.

Main Results:

• FACS increased oxidative stress, indicated by altered GSSG/GSH and NAD(P)H/NAD(P)+ ratios and a 50% rise in reactive oxygen species.
• Nearly half of profiled metabolomic features changed by at least 1.5-fold post-FACS.
• Significant alterations were observed in metabolites including glycogen, nucleosides, amino acids, central carbon metabolites, and acylcarnitines.
• Fetal bovine serum reduced but did not eliminate FACS-induced metabolic changes.

Conclusions:

• FACS significantly alters the metabolic state and induces oxidative stress in cells.
• Metabolomic data from FACS-sorted cells do not accurately reflect in vivo or pre-sorted physiological conditions.
• Buffer components and data normalization do not fully mitigate FACS-induced metabolic artifacts.