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Optimized protocol to isolate primary mouse peritoneal macrophage metabolites
Adam De Jesus1, Carolina M Pusec2, Tivoli Nguyen1
1Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Feinberg Cardiovascular Research Institute, Northwestern University, Chicago, IL 60611, USA.
STAR Protocols
|September 14, 2022
Summary
This study presents a new protocol for extracting metabolites from low numbers of primary peritoneal macrophages (PMs). This method enables crucial metabolomics analysis and stable isotope tracing in immune cells.
Area of Science:
- Immunology
- Metabolomics
- Cell Biology
Background:
- Peritoneal macrophages (PMs) exhibit superior stability compared to other macrophage subtypes.
- Limited cell numbers from single mice pose challenges for metabolomics analysis of PMs.
Purpose of the Study:
- To develop an efficient protocol for isolating metabolites from small quantities of mouse primary PMs.
- To facilitate 13C-stable glucose tracing and metabolomics analysis in low-cell-number PM samples.
Main Methods:
- A novel metabolite extraction protocol utilizing compound X, replacing traditional methanol-based methods.
- Application of the protocol to low cell number samples of primary mouse PMs.
- 13C-stable glucose tracing for metabolic pathway analysis.
Main Results:
- Consistent metabolite extraction from low cell number samples.
- Fewer procedural steps compared to conventional methanol-based extraction techniques.
- Successful application in metabolomics and stable isotope tracing.
Conclusions:
- The developed protocol effectively overcomes cell number limitations for PM metabolomics.
- This method provides a streamlined approach for analyzing macrophage metabolism.
- Enables advanced analyses like stable isotope tracing in scarce immune cell populations.
