Isotopic tracing of glucose metabolites in human monocytes to assess changes in inflammatory conditions

• Published in: STAR protocols Vol. 3; no. 4; p. 101715
• Main Authors: Giacomello, Ginevra, Böttcher, Chotima, Parr, Maria Kristina
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.12.2022; Elsevier
• Subjects: Chemistry; Glucose; Humans; Immunology; Mass Spectrometry; Metabolism; Metabolome; Metabolomics; Metabolomics - methods; Monocytes; Protocol; Metabolomics; Chemistry; Immunology; Metabolism; Mass spectrometry
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101715

Differences in metabolic profiles can link to functional changes of immune cells in disease conditions. Here, we detail a protocol for the detection and quantitation of 19 metabolites in one analytical run. We provide the parameters for chromatographic separation and mass spectrometric analysis of isotopically labeled and unlabeled metabolites. We include steps for incubation and sample preparation of PBMCs and monocytes. This protocol overcomes the chromatographic challenges caused by the chelating properties of some metabolites. [Display omitted] •Protocol for the quantitation of glucose metabolites•Protocol for the incubation and sample preparation of PBMCs and monocytes•Chromatographic separation of cell metabolites in major metabolic pathways•dMRM parameters for cell metabolites in major metabolic pathways Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Differences in metabolic profiles can link to functional changes of immune cells in disease conditions. Here, we detail a protocol for the detection and quantitation of 19 metabolites in one analytical run. We provide the parameters for chromatographic separation and mass spectrometric analysis of isotopically labeled and unlabeled metabolites. We include steps for incubation and sample preparation of PBMCs and monocytes. This protocol overcomes the chromatographic challenges caused by the chelating properties of some metabolites.