1. Probing Enzymatic Activity Inside Single Cells
- Author
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Aldo Jesorka, Shijun Xu, Owe Orwar, Stephen G. Weber, Ida Isaksson, Jessica Olofsson, Gavin D. M. Jeffries, and Helen Bridle
- Subjects
Cell Membrane Permeability ,01 natural sciences ,Fluorescence ,Article ,Analytical Chemistry ,Mice ,Neuroblastoma ,03 medical and health sciences ,chemistry.chemical_compound ,Cell Line, Tumor ,Animals ,Fluorescein ,030304 developmental biology ,0303 health sciences ,Chromatography ,Dose-Response Relationship, Drug ,biology ,Chemistry ,010401 analytical chemistry ,Substrate (chemistry) ,Enzyme assay ,Rats ,0104 chemical sciences ,Dose–response relationship ,Digitonin ,Membrane ,Levamisole ,Biochemistry ,biology.protein ,Alkaline phosphatase ,Single-Cell Analysis ,Intracellular - Abstract
We report a novel approach for determining the enzymatic activity within a single suspended cell. Using a steady-state microfluidic delivery device and timed exposure to the pore-forming agent digitonin, we controlled the plasma membrane permeation of individual NG108-15 cells. Mildly permeabilized cells (~100 pores) were exposed to a series of concentrations of fluorescein diphosphate (FDP), a fluorogenic alkaline phosphatase substrate, with and without levamisole, an alkaline phosphatase inhibitor. We generated quantitative estimates for intracellular enzyme activity and were able to construct both dose-response and dose-inhibition curves at the single-cell level, resulting in an apparent Michaelis contant Km of 15.3 μM ± 1.02 (mean ± standard error of the mean (SEM), n = 16) and an inhibition constant Ki of 0.59 mM ± 0.07 (mean ± SEM, n = 14). Enzymatic activity could be monitored just 40 s after permeabilization, and five point dose-inhibition curves could be obtained within 150 s. This rapid approach offers a new methodology for characterizing enzyme activity within single cells.
- Published
- 2013