Abstract
Activation of inflammasomes in peritoneal macrophages and intestinal epithelial cells (IEC) leads to the release of eicosanoids. To assess the amount of eicosanoids released by IEC, lipids need to be isolated from whole tissue previous to analysis by lipid mass spectrometry or ELISA. This protocol describes how to isolate lipids from intestinal tissue for analysis by PGE2-ELISA and normalize to tissue protein content.
Keywords: Intestine, Eicosanoid, ELISA, Prostaglandin, Lipid extraction
Background
Inflammasome induced eicosanoid release is a relatively recent observation. It is not clear which cell/tissue types other than peritoneal macrophages and intestinal epithelial cells (IEC) (Rauch et al., 2017) can release prostaglandins upon inflammasome activation yet. This protocol can be adapted for other types of tissue as well as measurement of eicosanoid release induced by other stimuli than inflammasome activation in intestinal tissue. Note that this protocol is specifically for use of eicosanoid analysis by ELISA, other protocols have been described for eicosanoid analysis by lipid mass spectrometry.
Materials and Reagents
Equipment
Procedure
Workflow:
Data analysis
Back-calculate sample PGE2 concentration according to the description in the ELISA protocol. Do not forget to account for taking only an aliquot of the spin supernatant. Calculate the amount of protein in sample according to BCA assay. Now you can calculate the ratio of PGE2/mg total protein in your sample.
Notes
Recipes
Acknowledgments
This protocol was developed with the help of the Gronert laboratory at UC Berkeley. Funding: HHMI and NIH grants (AI075039, AI063302, EY026082), Austrian Science Fund (FWF) (the Erwin Schroedinger Fellowship J3789-B22).
Competing interests
The author declares no conflict of interest.
Ethics
The animal experiments complied with the regulatory standards of, and were approved by, the University of California Berkeley Institutional Animal Care and Use Committee.
References
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