Chemical fixation to arrest phospholipid signaling for chemical cytometry

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This topic contains 0 replies, has 0 voices, and was last updated by  Devid Jhons 28th February 2018 at 9:42 am.

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  • #917

    Devid Jhons
    Participant

    Highlights


    High quality separation of phosphatidylinositol isomers was developed.

    Exogenously added fluorescent phospholipid is retained in cells after fixation.

    Fluorescent phospholipid was recovered from fixed cells.

    Fixation stops cellular reactions for later chemical cytometry of metabolites.

    Phospholipase C activity was measured in single, fixed leukemic cells.

    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 followed by glutaraldehyde fixation and immediate analysis, 24 ± 2% and 139 ± 12% of the lipid was recoverable, respectively, when compared to an unfixed control. Storage of the cells for 24 h yielded recoverable lipid of 61 ± 3% (C6-PIP2) and 55 ± 5% (C16-PIP2) when compared to cells analyzed immediately after fixation. The metabolites observed with and without fixation were identical. Measurement of phospholipase C activity in single leukemic cells in response to an agonist demonstrated the capability of chemical fixation coupled to single-cell CZE to yield an accurate snapshot of cellular reactions with the probe. This methodology enables cell assay with the reporter to be separated in space and time from reporter metabolite quantification while preserving assay integrity.

    #5164

    Mckiller
    Participant

    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. 

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