Capillary electrophoresis to analyse enzymatic pathways in natural killer cells

Webwriter Claire Weston @imperialcollege writes more…

Natural killer (NK) cells are a type of lymphocyte that are vitally important in the body’s immune response. These cells show high levels of heterogeneity, with an estimated 6000 to 30000 distinct subsets in the circulating blood of just one person. Because of this, technologies are needed that are capable of measuring single cells.

Allbritton and co-workers, at the University of North Carolina, have reported a method for doing just this using an automated single-cell capillary electrophoresis (CE) system. This system works by capturing individual NK cells in cell traps (consisting of 15µm diameter microwells) and positioning a capillary above one of the cell traps. The cell is lysed using a laser pulse and the cellular contents are injected into the capillary electrokinetically. The capillary is programmed to transfer the cellular contents to the electrophoretic buffer, where separation occurs. The capillary then moves back to the next cell trap and repeats the process.

The work in this paper focuses on the spingosine-1-phosphate (S1P) pathway (shown in the diagram below), which is important in the regulation of lymphocyte migration and differentiation, and cytokine production. Fluorescently labelled sphingosine was loaded into the NK cells of healthy human subjects and, following incubation, the cells were loaded onto the microwells and analysed using the single-cell CE system. The amount of each metabolite present was then identified from the electropherogram. The authors identified three major peaks, corresponding to fluorescently labelled sphingosine, S1P and hexadecanoic acid.  From the relative amounts of each metabolite, the activity of various enzymes in the S1P pathway were assessed.

The activity within the S1P pathway was found to be highly heterogeneous in NK cells obtained from one individual, as well as those from different subjects. In the majority of cells, phosphorylation of sphingosine was upregulated relative to the breakdown of S1P. No peaks were seen that corresponded to the ceramide metabolite, suggesting that in healthy humans, sphingosine is metabolised to S1P more rapidly.

By increasing the throughput of the automated system and preparing additional fluorescent reporters, this automated CE system has the potential to provide a more comprehensive picture of an individual cell’s signalling pathways.



To download the full article for free* click the link below:
Analysis of sphingosine kinase activity in single natural killer cells from peripheral blood
Alexandra J. DickinsonMegan MeyerErica A. PawlakShawn GomezIlona Jaspers and Nancy L. Allbritton
DOI: 10.1039/C5IB00007F


*Access is free until 31.05.2014 through a registered publishing personal account

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