TY - JOUR
T1 - An integrated toolbox to profile macrophage immunometabolism
AU - Verberk, Sanne G. S.
AU - de Goede, Kyra E.
AU - Gorki, Friederike S.
AU - van Dierendonck, Xanthe A. M. H.
AU - Argüello, Rafael J.
AU - van den Bossche, Jan
N1 - Funding Information: We thank Sam de Decker, Enrico Tatti, and Barry Bochner for supplying all materials to perform the Biolog mitochondrial-substrate-coated and carbon-substrate-coated assays, their help with setting up the assays and interpreting the results, and their helpful comments on the manuscript. We further thank Amber Eggert for optimizing the Hoechst cell-counting protocol on the Cytation five and Karl Harber for proofreading the manuscript. We thank ANR and Inserm Transfert for the ANR-20-CE-CE14-0028-01 and CoPoC MAT-PI-17493-A-04 grants to R.J.A. J.V.d.B. received a Netherlands Heart Foundation senior fellowship (2017T048). Additionally, J.V.d.B. received an Enw-Klein-1 grant from NWO (OCENW.KLEIN.268) and a consortia grant from European Research Area network on Cardiovascular Diseases (ERA-CVD 2019T108). Last, we acknowledge support from Cancer Center Amsterdam. S.V. and K.d.G. contributed equally to this work; order of authorship was decided by flipping a coin. Conceptualization, J.V.d.B. S.V. and K.d.G; methodology, S.V. K.d.G. F.G. and R.A.; formal analysis, S.V. and K.d.G.; investigation, S.V. K.d.G. F.G. and X.v.D.; visualization, S.V. and K.d.G.; writing ? review & editing, S.V. K.d.G. F.G. X.v.D. R.A. and J.V.d.B.; funding acquisition, J.V.d.B. All authors read and approved the final manuscript. The authors declare no competing interests. There are restrictions to the commercial use of SCENITH due to a pending patent application (PCT/EP2020/060,486). Funding Information: We thank Sam de Decker, Enrico Tatti, and Barry Bochner for supplying all materials to perform the Biolog mitochondrial-substrate-coated and carbon-substrate-coated assays, their help with setting up the assays and interpreting the results, and their helpful comments on the manuscript. We further thank Amber Eggert for optimizing the Hoechst cell-counting protocol on the Cytation five and Karl Harber for proofreading the manuscript. We thank ANR and Inserm Transfert for the ANR-20-CE-CE14-0028-01 and CoPoC MAT-PI-17493-A-04 grants to R.J.A. J.V.d.B. received a Netherlands Heart Foundation senior fellowship ( 2017T048 ). Additionally, J.V.d.B. received an Enw-Klein-1 grant from NWO ( OCENW.KLEIN.268 ) and a consortia grant from European Research Area network on Cardiovascular Diseases ( ERA-CVD 2019T108 ). Last, we acknowledge support from Cancer Center Amsterdam . Publisher Copyright: © 2022 The Author(s)
PY - 2022/4/25
Y1 - 2022/4/25
N2 - Macrophages are dynamic immune cells that can adopt several activation states. Fundamental to these functional activation states is the regulation of cellular metabolic processes. Especially in mice, metabolic alterations underlying pro-inflammatory or homeostatic phenotypes have been assessed using various techniques. However, researchers new to the field may encounter ambiguity in choosing which combination of techniques is best suited to profile immunometabolism. To address this need, we have developed a toolbox to assess cellular metabolism in a semi-high-throughput 96-well-plate-based format. Application of the toolbox to activated mouse and human macrophages enables fast metabolic pre-screening and robust measurement of extracellular fluxes, mitochondrial mass and membrane potential, and glucose and lipid uptake. Moreover, we propose an application of SCENITH technology for ex vivo metabolic profiling. We validate established activation-induced metabolic rewiring in mouse macrophages and report new insights into human macrophage metabolism. By thoroughly discussing each technique, we hope to guide readers with practical workflows for investigating immunometabolism.
AB - Macrophages are dynamic immune cells that can adopt several activation states. Fundamental to these functional activation states is the regulation of cellular metabolic processes. Especially in mice, metabolic alterations underlying pro-inflammatory or homeostatic phenotypes have been assessed using various techniques. However, researchers new to the field may encounter ambiguity in choosing which combination of techniques is best suited to profile immunometabolism. To address this need, we have developed a toolbox to assess cellular metabolism in a semi-high-throughput 96-well-plate-based format. Application of the toolbox to activated mouse and human macrophages enables fast metabolic pre-screening and robust measurement of extracellular fluxes, mitochondrial mass and membrane potential, and glucose and lipid uptake. Moreover, we propose an application of SCENITH technology for ex vivo metabolic profiling. We validate established activation-induced metabolic rewiring in mouse macrophages and report new insights into human macrophage metabolism. By thoroughly discussing each technique, we hope to guide readers with practical workflows for investigating immunometabolism.
KW - immunometabolism
KW - macrophages
KW - metabolism
KW - semi-high throughput screening
KW - toolbox
UR - http://www.scopus.com/inward/record.url?scp=85129149260&partnerID=8YFLogxK
U2 - https://doi.org/10.1016/j.crmeth.2022.100192
DO - https://doi.org/10.1016/j.crmeth.2022.100192
M3 - Article
C2 - 35497494
SN - 2667-2375
VL - 2
JO - Cell Reports Methods
JF - Cell Reports Methods
IS - 4
M1 - 100192
ER -