Optimized protocol to isolate primary mouse peritoneal macrophage metabolites

• Published in: STAR protocols Vol. 3; no. 4; p. 101668
• Main Authors: De Jesus, Adam, Pusec, Carolina M., Nguyen, Tivoli, Keyhani-Nejad, Farnaz, Gao, Peng, Weinberg, Samuel E., Ardehali, Hossein
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.12.2022; Elsevier
• Subjects: Animals; Cell isolation; Glucose; Immunology; Macrophages, Peritoneal; Mass spectrometry; Metabolism; Metabolomics; Metabolomics - methods; Methanol; Mice; Protocol; Metabolomics; Immunology; Metabolism; Cell isolation; Mass spectrometry
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101668

Peritoneal macrophages (PMs) have been shown to have higher stability compared to other macrophage subtypes. However, obtaining enough PMs from a single mouse is often a limitation for metabolomics analysis. Here, we describe a protocol to isolate metabolites from a small number of mouse primary PMs for 13C-stable glucose tracing and metabolomics. Our protocol uses X for metabolite extraction instead of methanol. Our protocol can consistently extract metabolites from low cell number samples with fewer steps than methanol-based approaches. For complete details on the use and execution of this protocol, please refer to De Jesus et al., (2022). [Display omitted] •Streamlined isolation of murine peritoneal macrophages•Efficient metabolite extraction of limited sample amount•Detection of carbon-labeled polar metabolites from small sample size•Protocol applied to mouse primary PMs, but applicable to other cell types Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Peritoneal macrophages (PMs) have been shown to have higher stability compared to other macrophage subtypes. However, obtaining enough PMs from a single mouse is often a limitation for metabolomics analysis. Here, we describe a protocol to isolate metabolites from a small number of mouse primary PMs for 13C-stable glucose tracing and metabolomics. Our protocol uses X for metabolite extraction instead of methanol. Our protocol can consistently extract metabolites from low cell number samples with fewer steps than methanol-based approaches.