Flow Cytometry Has a Significant Impact on the Cellular Metabolome

Area of Science:

• Immunology
• Cell Biology
• Biochemistry

Background:

• Characterizing specialized cell subpopulations is crucial for understanding organ function in health and disease.
• Fluorescence-activated cell sorting (FACS) is a common technique for isolating cells based on marker expression.

Purpose of the Study:

• To investigate the impact of FACS on the cellular metabolome of mouse peritoneal macrophages.
• To identify FACS-induced biochemical alterations in macrophages.

Main Methods:

• Mouse peritoneal macrophages were subjected to FACS.
• The metabolome of FACS-treated macrophages was compared to directly pelleted macrophages using mass spectrometry-based techniques.
• Analysis focused on metabolites related to cellular stress, energy consumption, and cell damage.

Main Results:

• FACS treatment led to altered metabolite profiles in macrophages compared to controls.
• Changes were observed in metabolites associated with the plasma membrane, indicating mechanosensory signaling activation and inflammation-like stress.
• FACS also induced alterations in energy metabolism and markers of cell damage, primarily due to physical stress from instrument passage.

Conclusions:

• FACS induces significant biochemical changes in mouse peritoneal macrophages.
• These FACS-induced metabolic alterations, stemming from physical cell manipulation, can impact experimental outcomes.
• Researchers should account for these biochemical changes when designing metabolic assays using flow cytometry-sorted cells.