Chemical fixation to arrest phospholipid signaling for chemical cytometry

Journal of Chromatography. a
Angela ProctorN L Allbritton

Abstract

Chemical cytometry is a powerful tool for measuring biological processes such as enzymatic signaling at the single cell level. Among these technologies, single-cell capillary zone electrophoresis (CZE) has emerged as a powerful tool to assay a wide range of cellular metabolites. However, analysis of dynamic processes within cells remains challenging as signaling pathways are rapidly altered in response to changes in the cellular environment, including cell manipulation and storage. To address these limitations, we describe a method for chemical fixation of cells to stop the cellular reactions to preserve the integrity of key signaling molecules or reporters within the cell and to enable the cell to act as a storage reservoir for the reporter and its metabolites prior to assay by single-cell CZE. Fluorescent phosphatidylinositol 4,5-bisphosphate reporters were loaded into cells and the cells were chemically fixed and stored prior to analysis. The reporter and its metabolites were electrophoretically separated by single-cell CZE. Chemical fixation parameters such as fixative, fixation time, storage solution, storage duration, and extraction solution were optimized. When cells were loaded with a fluorescent C6- or C16-PIP2 followe...Continue Reading

Citations

Dec 26, 2018·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Kellen DeLaneyLingjun Li
Oct 13, 2020·Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry·Takayuki Kawai
Mar 11, 2020·Trends in Analytical Chemistry : TRAC·Luke A GallionNancy L Allbritton
Jan 30, 2018·ACS Chemical Biology·Brianna M VickermanDavid S Lawrence

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