Abstract
Here, we report on a novel protocol for determining the viability of individual cells in an adherent cell culture, without adversely affecting the remaining cells in the sample. This is facilitated using a freestanding microfluidic perfusion device, the Multifunctional Pipette (MFP), which generates a virtual flow cell around selected single cells. We investigated the utility on four different cell lines, NG108-15, HEK 293, PC12, and CHO, and combined the assay with a cell poration experiment, in which we apply the pore-forming agent digitonin, followed by fluorescein diphosphate, a pre-fluorescent substrate for alkaline phosphatase, in order to monitor intracellular enzyme activity. The cell viability was instantly assessed through simultaneous perfusion with fluorescein diacetate (FDA) and propidium iodide (PI), both being dispensed through the same superfusion device used to porate and deliver the enzyme substrate. In this fluorescence assay, viable and non-viable cells were distinguished by their green and red emission, respectively, within 10 s. In addition, the enzyme activity was monitored over time as a secondary test for cellular activity. Our findings demonstrate that this microfluidic technology-assisted approach is a facile, rapid, and reliable means to determine the viability in single-cell experiments and that viability studies can be performed routinely alongside typical substrate delivery protocols. This approach would remove the need for global cell viability testing and would enable viability studies of only the cells under experimental analysis.
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Acknowledgments
We thank Prof. Owe Orwar for his support. This research was funded by the European Research Council (ERC), the Chalmers Area of Advance in Nanoscience and Technology, The Nordic Council of Ministers (Nordforsk), and the Swedish Research Council (VR).
Conflict of interest
Gavin D. M. Jeffries and Aldo Jesorka are both founders of Fluicell AB. No payments or financial gain was a reason for, or a direct consequence of, the research contained within the manuscript.
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Xu, S., Kim, A., Jeffries, G.D.M. et al. A rapid microfluidic technique for integrated viability determination of adherent single cells. Anal Bioanal Chem 407, 1295–1301 (2015). https://doi.org/10.1007/s00216-014-8364-9
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DOI: https://doi.org/10.1007/s00216-014-8364-9