Monitoring the cellular activity of a cultured single cell by scanning electrochemical microscopy (SECM). A comparison with fluorescence viability monitoring
Introduction
The activity of living cells is significantly influenced by chemical and physical stimuli; therefore, it is important to measure the cellular activity during environmental monitoring and drug screening. There are various methods for monitoring the change in the physiology of cultured cells, e.g. the rate of uptake of glucose and oxygen (Karube et al., 1982, Li et al., 1988), the production of heat (Hammerstedt and Lovrein, 1983, James, 1987), and pH monitoring (McConnell et al., 1992, Hafner, 2000). The microphysiometer based on pH monitoring has attracted attention as a relatively new approach of drug screening of cultured living cells.
The fluorescence imaging of living cells has become popular since it provides real-time information on the cellular status at a single-cell level. There have been many types of fluorescent stains that interact with intracellular species to emit fluorescence, and some stains such as Calcein-AM and propidium iodide (PI) have been used for imaging the viability of cells and bacteria (Comas and Vives-Rego, 1998, Hodder et al., 2000, Noël-Hudson et al., 1997, Roden et al., 1999). Such a fluorescent measurement is, however, unsuitable for long-term measurement because of fluorescent fading of the staining reagents by photochemical decomposition.
Metabolic activity is an important factor to estimate cellular activity. Among the various analytical tools for monitoring metabolic activity, scanning electrochemical microscopy (SECM) has received attention since it is capable of imaging metabolic activity at the single cell level. Berger et al. investigated the activity of osteclasts using SECM as a tool to detect Ca2+ (Berger et al., 2001). Liu et al. used SECM for monitoring the enzymatic redox activity of human breast cells (Liu et al., 2000). Our group previously reported the possible use of SECM as a noninvasive tool to monitor the cellular status, in which the tip of a microelectrode is scanned near the cells to map the localized distribution of electroactive species such as oxygen (Nishizawa et al., 2002, Yasukawa et al., 1998, Yasukawa et al., 1999a, Yasukawa et al., 1999b, Yasukawa et al., 2000, Shiku et al., 2001).
We now report the use of the SECM as a tool to evaluate the effect of chemicals on the cellular status compared with the conventional fluorescence measurements. The time courses of the cellular activity of cultured HeLa cells are measured during exposure to KCN, ethyl alcohol, and the antibiotic drug, Antimycin A. It is clearly shown that the SECM-based activity monitoring is particularly suitable for detecting the effect of a respiratory inhibitor. The invasive nature of the SECM assay enables continuous monitoring of the dose response, which is important in the field of biomedical drug screening.
Section snippets
Materials and methods
KCN, ethyl alcohol and Antimycin A were purchased from Wako Pure Chemicals and used without further purification. Calcein-AM was purchased from Molecular Probes and used for the fluorescence imaging. All the solutions were prepared using distilled and deionized water.
The HeLa S3 cell line was donated by the Cell Resource Center for Biomedical Research Institute of Development (Tohoku University) and cultured on polystyrene dishes or glass-bottom dishes in RPMI1640 medium (Gibco) containing 10%
Monitoring cellular activity by fluorescence and SECM imaging upon exposure to KCN and ethyl alcohol
Fig. 2 shows changes in the fluorescence (a–c) and SECM (d–e) images of HeLa cells after exposure to 20 mM KCN. Before the addition, all the HeLa cells show a strong fluorescent emission (image (a)), indicating all the cells were in live state. Upon replacing the buffer solution with the 20 mM KCN, the Calcein fluorescence intensity of individual cells gradually decreased and eventually disappeared (images (b) and (c)). The change in the fluorescence intensity corresponds to the cellular
Conclusion
In the present study, we have applied SECM and fluorescence to measure the activity change in a single HeLa cell exposed to drugs. We clearly showed that the SECM measurement was very suitable for the screening of drugs that act on the respiration activity, such as KCN and Antimycin A. This is in contrast to the fact that the fluorescence measurement using Calcein is suitable to detect the drug action influencing the cellular membrane condition. The combination of the SECM with fluorescent
Acknowledgements
The present work was supported by Grants-in-Aid for Scientific Research on Priority Area (single cell molecular technology, No. 1122720) and B (No. 13450348) from the Ministry of Education, Science and Culture, Japan.
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