Anal. Chem. 2007, 79, 9414-9419
Analysis of Intracellular Oxygen and Metabolic Responses of Mammalian Cells by Time-Resolved Fluorometry Toma´s C. O’Riordan,† Alexander V. Zhdanov,‡ Gelii V. Ponomarev,§ and Dmitri B. Papkovsky*,‡
Luxcel Biosciences Ltd., BioInnovation Centre, UCC, Cork, Ireland, Biochemistry Department, University College Cork, Cavanagh Pharmacy Building, Cork, Ireland, and Institute of Biomedical Chemistry, Pogodinskaia Street 10, Moscow 119992, Russia
A simple, minimally invasive methodology for the analysis of intracellular oxygen in populations of live mammalian cells is described. Loading of the cells with the phosphorescent O2-sensing probe, MitoXpress, is achieved by passive liposomal transfer or facilitated endocytosis, followed by monitoring in standard microwell plates on a time-resolved fluorescent reader. Phosphorescence lifetime measurements provide accurate, real-time, quantitative assessment of average oxygen levels in resting cells and their alterations in response to stimulation. Analytical performance of the method is examined, optimized, and then demonstrated with different suspension and adherent cell lines including Jurkat, PC12, A549, HeLa, SHSY5Y, and C2C12, by monitoring responses to mitochondrial uncouplers, inhibitors, plasma membrane depolarization, and Ca2+ effectors. The assay provides relevant, information-rich data on cellular function and metabolism. It allows monitoring of small, rapid, and transient changes in cell respiration and screening of new chemical entities. As the terminal acceptor of the electron transport electron transport chain and oxidative phosphorylation, molecular oxygen is of paramount importance for the functioning of aerobic cells and organisms.1 Accordingly, O2 concentration and consumption rate are seen as fundamental markers of cell viability and metabolic status. The information gained from respirometric studies can be used to elucidate mitochondrial function,2 biochemical and signaling pathways,3-5 effects of different stimuli,6 and disease states,1,7 or to screen for new drugs.8 A number of * To whom correspondence should be addressed. Phone: +353-21-4901698. E-mail:
[email protected]. † Luxcel Biosciences Ltd. ‡ University College Cork. § Institute of Biomedical Chemistry. (1) Duchen, M. R. Mol. Aspects Med. 2004, 25, 365-451. (2) Marroquin, L. D.; Hynes, J.; Dykens, J. A.; Jamieson, J. D.; Will, Y. Toxicol. Sci. 2007, 97, 539-547. (3) Moncada, S.; Erusalimsky, J. D. Nat. Rev. Mol. Cell Biol. 2002, 3, 214220. (4) Nicholls, D. G. Cell Calcium 2005, 38, 311-317. (5) Kirichok, Y.; Krapivinsky, G.; Clapham, D. E. Nature 2004, 427, 360-364. (6) Hagen, T.; Taylor, C. T.; Lam, F.; Moncada, S. Science 2003, 302, 19751978. (7) Abramov, A. Y.; Scorziello, A.; Duchen, M. R. J. Neurosci. 2007, 27, 11291138.
9414 Analytical Chemistry, Vol. 79, No. 24, December 15, 2007
techniques are available for measurement of O2 consumption in samples containing cells, isolated mitochondria, tissue, and small organisms, particularly using extracellular O2 sensors and probes.9,10 Currently, fine parameters such as availability, distribution, and local O2 gradients within the cell are of high interest as they can provide a more detailed insight into processes taking place in the cell. However, unlike other parameters of cellular function such as Ca2+, ATP, MMP, and ROS, intracellular oxygen (icO2) is currently not analyzed routinely, which is due to the lack of appropriate measurement techniques and probes. Microelectrodes have been employed to measure icO2,11 but their invasive and consumptive nature is disadvantageous. Sensing of O2 by fluorescence quenching, a nonchemical, reversible (collisional) process, circumvents these problems and provides means for contactless measurement of O2.12 A number of different probe chemistries and techniques for sensing and imaging O2 in live cells have been described;12-15 however, their use to date has been limited due to their complexity or the need of sophisticated and highly specialized equipment. One of the recently developed imaging methods employs macromolecular (dye-protein conjugates), near-infrared phosphorescent probes, which are passively loaded into the cell cytoplasm and then retained there and “sense” icO2. This allows real-time analysis of icO2 in individual mammalian cells with high spatial and temporal resolution, monitoring responses to respiratory effectors.12 Notwithstanding these advances, live-cell fluorescence imaging remains a relatively complex, expensive technique with limited sample throughput. There (8) Papkovsky, D. B.; Hynes, J.; Will, Y. Exp. Opin. Drug Metab. Toxicol. 2006, 2. (9) Will, Y.; Hynes, J.; Ogurtsov, V. I.; Papkovsky, D. B. Nat. Protoc. 2006, 1, 2563-2572. (10) Wu, M.; Neilson, A.; Swift, A. L.; Moran, R.; Tamagnine, J.; Parslow, D.; Armistead, S.; Lemire, K.; Orrell, J.; Teich, J.; Chomicz, S.; Ferrick, D. A. Am. J. Physiol. Cell. Physiol. 2007, 292, C125-C136. (11) Cringle, S. J.; Yu, P. K.; Su, E. N.; Yu, D. Y. Invest. Ophthalmol. Vis. Sci. 2006, 47, 4072-4076. (12) O’Riordan, T. C.; Fitzgerald, K.; Ponomarev, G. V.; Mackrill, J.; Hynes, J.; Taylor, C.; Papkovsky, D. B. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2007, 292, R1613-R1620. (13) Schmalzlin, E.; van Dongen, J. T.; Klimant, I.; Marmodee, B.; Steup, M.; Fisahn, J.; Geigenberger, P.; Lohmannsroben, H. G. Biophys. J. 2005, 89, 1339-1345. (14) Koo, Y. E.; Cao, Y.; Kopelman, R.; Koo, S. M.; Brasuel, M.; Philbert, M. A. Anal. Chem. 2004, 76, 2498-2505. (15) Hogan, M. C. J. Appl. Phys. 1999, 86, 720-724. 10.1021/ac701770b CCC: $37.00
© 2007 American Chemical Society Published on Web 11/15/2007
is a need for more simple screening techniques for icO2 that are suitable for routine use. Here we describe one such assay system for monitoring icO2 in cell populations, which operates in a convenient microwell plate format with time-resolved fluorescence (TR-F) detection. The assay was designed for use with commercially available reagents and instrumentation, optimized, and demonstrated with different cells and respiratory effectors. EXPERIMENTAL SECTION Methods. Preparation and Loading of the Cells. HeLa, A549, Jurkat, SH-SY5Y, C2C12, and PC12 cells were obtained from ATCC. Suspension Jurkat cells were cultured to a concentration of ∼106 cells/mL in 75-cm2 flasks (Sarstedt) in RPMI medium supplemented with 10% fetal bovine serum (FBS), 2 mM Lglutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin (all Sigma), followed by centrifugation and resuspension in the same volume of medium. For loading, the O2-sensing probe based on the phosphorescent Pt-coproporphyrin dye conjugated to serum albumin16 (available under the trade name of MitoXpress from Luxcel Biosciences, Cork, Ireland) was reconstituted in 0.1 mL of medium to give 10 µM stock. The 1-mL aliquots of cell suspension were transferred to each well of a six-well plate (Sarstedt), to which 100 µL of probe stock (final concentration
