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Analyzing macrophage metabolism with Met-Flow

In this application note, Dr Graham Heieis from Leiden University Medical Center analyzes macrophage metabolism using Met-Flow with a selection of Abcam antibodies that target various core metabolic proteins.

Published 31st March 2022

Download the app note here.

Cellular metabolism is central to the survival and function of any living cell. Yet, its contribution in controlling the behavior of immune cells has become widely appreciated much more recently. Immune cells must be able to respond rapidly and diversely to combat the wide array of infectious threats to the host. It is now clear that changes in intrinsic metabolic activity are an underlying facet of this functional flexibility1,2. As intense research in immunometabolism within the past decade has provided important insights into disease mechanisms, it has also yielded great therapeutic potential, particularly in regards to tumor immunotherapy3. Given the importance of metabolism in immunity, and its promise to develop new treatments, it is increasingly becoming an integrated part of immunological studies.

Several technologies have been applied to investigate immune cells' metabolic requirements; however, as studies progress deeper into our understanding of immunometabolism, the technologies become more limited. Commonly used Seahorse XF analysis provides detailed real-time measurements of metabolic activity but requires an abundance of cells that is often not achievable, especially in the single-cell era. A preliminary picture of cellular metabolism can be obtained by single-cell RNA-sequencing but faces the caveats of limited read depth and discordance between transcription and translation, which was previously shown for metabolic enzymes in immune cells5.

Flow-cytometric methods so far have found a compromise for overcoming some of these limitations. Recent studies on T cells have used both standard flow cytometry6 or mass cytometry7,8 to validate the detection of enzymatic targets as a metabolic readout in vitro and ex vivo, matching changes in metabolism and protein expression to the cell functional status. Here, we further validate if a single-cell metabolic analysis strategy called Met-Flow6 can distinguish the metabolic status of immune cells using in vitro differentiated macrophages.

Macrophages are seeded throughout all tissues in the body and carry out vital functions to maintain tissue homeostasis and protect against pathogens. The metabolism of macrophages has been well studied in vitro, using the classical "M1" or "M2" models that represent the extremes of macrophage activation. In this system, M1 inflammatory macrophages, associated with intracellular infection, adopt a highly glycolytic metabolism9. In contrast, alternatively activated M2 macrophages, normally associated with helminth infection or tissue wound-healing, acquire a dominantly mitochondrial metabolism that uses glutaminolysis and fatty acid oxidation to sustain OXPHOS10,11. Here we assess macrophage metabolism using Met-Flow with a selection of Abcam antibodies that target various core metabolic proteins. We confirm that the selected antibodies elucidate metabolic differences between in vitro differentiated bone-marrow macrophages and can also distinguish murine immune populations directly ex vivo. This method opens the door for further interrogation of metabolism using the vast array of metabolic targets available through Abcam with flexible panel design and further analysis of physiological samples from rodents or humans. 

Download the pdf to see the data.

Metabolism is an extensive network of transporters and enzymes, but early work in immuno-metabolism focused on a handful of central core pathways used to generate energy for the cell (Fig. 1). These pathways were glycolysis, oxidative phosphorylation (OXPHOS), glutaminolysis, long-chained fatty acid oxidation and synthesis, and the pentose phosphate pathway (PPP)1. Depending on the activation, location or effector state of the cell, these pathways were found to be dynamically regulated, particularly at the branch point of aerobic glycolysis and mitochondrial oxidation. Glycolysis has been linked more strongly to cells in an inflammatory or acute effector state, such as recently activated T cells. In contrast, mitochondrial OXPHOS is associated more with cells possessing a regulatory phenotype or are long-lived, including memory and regulatory T cells1,4.Cellular metabolism is central to the survival and function of any living cell. Yet, its
contribution in controlling the behavior of immune cells has become widely appreciated
much more recently. Immune cells must be able to respond rapidly and diversely to
combat the wide array of infectious threats to the host. It is now clear that changes
in intrinsic metabolic activity are an underlying facet of this functional flexibility1,2. As
intense research in immuno-metabolism within the past decade has provided important
insights into disease mechanisms, it has also yielded great therapeutic potential,
particularly in regards to tumor immunotherapy3. Given the importance of metabolism
in immunity, and its promise to develop new treatments, it is increasingly becoming an
integrated part of immunological studies.
Metabolism is an extensive network of transporters and enzymes, but early work in
immuno-metabolism focused on a handful of central core pathways used to generate
energy for the cell (Fig. 1). These pathways were glycolysis, oxidative phosphorylation
(OXPHOS), glutaminolysis, long-chained fatty acid oxidation and synthesis, and the
pentose phosphate pathway (PPP)1. Depending on the activation, location or effector
state of the cell, these pathways were found to be dynamically regulated, particularly
at the branch point of aerobic glycolysis and mitochondrial oxidation. Glycolysis has
been linked more strongly to cells in an inflammatory or acute effector state, such
as recently activated T cells. In contrast, mitochondrial OXPHOS is associated more
with cells possessing a regulatory phenotype or are long-lived, including memory and
regulatory T cells1,4.Cellular metabolism is central to the survival and function of any living cell. Yet, its
contribution in controlling the behavior of immune cells has become widely appreciated
much more recently. Immune cells must be able to respond rapidly and diversely to
combat the wide array of infectious threats to the host. It is now clear that changes
in intrinsic metabolic activity are an underlying facet of this functional flexibility1,2. As
intense research in immuno-metabolism within the past decade has provided important
insights into disease mechanisms, it has also yielded great therapeutic potential,
particularly in regards to tumor immunotherapy3. Given the importance of metabolism
in immunity, and its promise to develop new treatments, it is increasingly becoming an
integrated part of immunological studies.
Metabolism is an extensive network of transporters and enzymes, but early work in
immuno-metabolism focused on a handful of central core pathways used to generate
energy for the cell (Fig. 1). These pathways were glycolysis, oxidative phosphorylation
(OXPHOS), glutaminolysis, long-chained fatty acid oxidation and synthesis, and the
pentose phosphate pathway (PPP)1. Depending on the activation, location or effector
state of the cell, these pathways were found to be dynamically regulated, particularly
at the branch point of aerobic glycolysis and mitochondrial oxidation. Glycolysis has
been linked more strongly to cells in an inflammatory or acute effector state, such
as recently activated T cells. In contrast, mitochondrial OXPHOS is associated more
with cells possessing a regulatory phenotype or are long-lived, including memory and
regulatory T cells1,4.