Toxicology in Vitro 21 (2007) 176–182 www.elsevier.com/locate/toxinvit
Cytomics: A multiparametric, dynamic approach to cell research Guadalupe Herrera, Laura Diaz, Alicia Martinez-Romero, Angela Gomes, Eva Villamón, Robert C. Callaghan, José-Enrique O’Connor ¤ Laboratorio de Citómica, Unidad Mixta de Investigación CIPF-UVEG, Centro de Investigación Príncipe Felipe, Avda. Autopista del Saler, 16, 46013 Valencia, Spain Received 8 April 2006; accepted 13 July 2006 Available online 22 July 2006
Abstract Cytomics aims to determine the molecular phenotype of single cells. Within the context of the -omics, cytomics allows the investigation of multiple biochemical features of the heterogeneous cellular systems known as the cytomes. Cytomics can be considered as the science of single cell-based analyses that links genomics and proteomics with the dynamics of cell and tissue function, as modulated by external inXuences. Inherent to cytomics are the use of sensitive, scarcely invasive, Xuorescence-based multiparametric methods and the event-integrating concept of individual cells to understand the complexity and behaviour of tissues and organisms. Among cytomic technologies, Xow cytometry, confocal laser scanning microscopy and laser capture microdissection are of great relevance. Other recent technologies based on single cell bioimaging and bioinformatic tools become important in drug discovery and toxicity testing, because of both highcontent and high-troughput. The multiparametric capacity of cytomics is very useful for the identiWcation, characterization and isolation of stem cell populations. In our experience, Xow cytometry is a powerful and versatile tool that allows quantitative analysis of single molecules, prokaryotic and eukaryotic cells for basic, biotechnological, environmental and clinical studies. The dynamic nature of cytomic assays leads to a real-time kinetic approach based on sequential examination of diVerent single cells from a population undergoing a dynamic process, the in Xuxo level. Finally, cytomic technologies may provide in vitro methods alternative to laboratory animals for toxicity assessment. © 2006 Elsevier Ltd. All rights reserved. Keywords: Cytometry; Fluorescence; Toxicology; Pharmacology; In vitro
1. Cytomics and cytomes: single-cell based analysis in complex systems Genomics, proteomics and metabolomics provide outstanding technical contributions to Cell Biology but become limited when single or scarce cells are examined or fast cellular processes followed kinetically (Figeys, 2004). In addition, the importance of epigenetic changes (Plass, 2002) and the modulatory inXuence of cell environment (Abrous et al., 2005) recommend to combine “-omic” studies with functional analysis (Bernas et al., 2006). Because of this, cell-based assays are increasingly sought for basic research
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and drug screening, since they approach complex cell-tocell and cell-to-environment interactions, in assay formats that are both information-rich and technically convenient. In this context, where “-omics” meet Systems Biology, cytomics represents a novel analytical strategy aimed to determine multiple biochemical features (the molecular phenotype) in single cells and may be deWned as the cytometry of complex cellular systems. In analogy with other -omics (genomics/genome; proteomics/proteome, and so on) the objective of cytomics are the heterogeneous cellular systems known as the cytomes. The cytomes can be understood as the heterogeneous cellular systems and functional components of the pluricellular organisms. Since the functional heterogeneity of the cytomes results from both the genome and extracellular environment, cytomics can be considered as a discipline that
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links genomics and proteomics to cell and tissue function, as modulated by external inXuences. Of special importance is the cell-by-cell basis of cytomic analysis, an approach that allows to resolve heterogeneous systems and avoids the loss of information that characterizes bulk technologies, in which average values are obtained from large number of cells or from tissue homogenates. For a deeper, comprehensive view of cytomics and their relevance to biomedicine, see Valet (2005) and Bernas et al. (2006). 2. Technical and analytical features of cytomic technologies Inherent to cytomics are the use of sensitive, scarcely invasive, Xuorescence-based methods and the integrating concept of individual cell analysis to understand the complexity and behaviour of tissues and organisms. Due to the availability of large number of Xuorescent markers and the multiplicity of Xuorescence detectors interfaced to the dedicated instrumentation, cytomic assays may be multiparametric, polychromatic and multiplexed. Fluorescence-based measurements may be qualitative and quantitative and can be obtained as the result of single end-point measurement or kinetic, sequential measurements. While these features are common to all cytomic technologies, there are important speciWc diVerences depending on whether the quantitative cell Xuorescence data are extracted together with cell morphology in image-based cytomics (Eils and Athale, 2003) or from Xuorescence-pulse analysis in Xow-based cytomics (O’Connor et al., 2001). Among the current cytomic technologies, Xow cytometry (FCM), confocal laser scanning microscopy (CLSM), spinning-disk confocal microscopy (SDCM) and laser scanning cytometry (LSC) are of established relevance. Other cytomic technologies based on single-cell based image analysis and powerful bioinformatic tools (high-content screening bioimaging, HCSB) have been recently introduced for drug discovery and toxicity testing, as they can provide both high-content and high-troughput analysis. Finally, laser capture microdissection (LCM), a preparative technique for obtaining pure cells from speciWc microscopic regions of tissue sections, can also be considered among cytomic technologies.
susceptibility or resistance. The advantages of FCM derive from its multiparametricity that provides multiple, simultaneous targets to assess cell lesion or death in selected cell populations, either as end-point or kinetic measurements. 2.2. Confocal Xuorescence microscopy Confocal Xuorescence microscopy is advantageous to conventional Xuorescence microscopy, as it narrows the Weld depth, eliminates out-of-focus blur and allows to produce serial optical stacks from thick specimens (z-axis resolution). Confocality may be applied to image single cells from Wxed or living preparations labelled with appropriate Xuorescent probes. This technique allows sophisticated cell information based on spatial- and time-resolved Xuorescence measurements and as such is being increasingly used for scientiWc and technological applications (Rubart, 2004; Ivorra et al., 2006). Usually, CLSM yields better image quality but the imaging frame rate is slow while SDCM may produce video rate imaging, which is required for eYcient dynamic observations (Maddox et al., 2003). However, the new confocal systems function like hybrids allowing to obtain both high speed and good resolution. 2.3. Laser scanning cytometry LSC is a microscope-based, scanning cytoXuorimeter that combines the advantages of Xow and image cytometry. It allows multiparametric analysis performed directly by measuring the Xuorescence of individual cells in solid-state preparations, such as monolayers, smears, imprints, cytospins or tissue sections in several supports including slides, dishes and multiwell plates. LSC provides increased sensitivity and speciWcity compared to traditional microscopic techniques and a similar structure for data analysis to Xow cytometry, albeit at lower data acquisition velocity. In addition, it allows relocation of the coordinates of analyzed cells of interest following slide restaining. Finally, single cells identiWed by their Xuorescence measurements can be individualized from histogram or dot-plot displays and shown as single-cell pictures or combined in picture galleries for further analysis (Juan and Cordon-Cardo, 2001).
2.1. Flow cytometry
2.4. High-content screening bioimaging
This methodology requires that cells (or microscopical biological particles) are in suspension. FCM allows the simultaneous quantiWcation of multiple Xuorescence emissions in the same cell, arising from Xuorescent markers, and scattered light related to morphology, revealing key cellular functions or structures (O’Connor et al., 2001). The velocity of analysis can be up to thousands of single-cells per second and individual cells from heterogeneous subpopulations can be physically isolated on the basis of their Xuorescence or light scatter properties. The multiparametric capacity of FCM permits to quantify the eVects induced by the exposure to a toxic agent, providing a direct proof of cellular
An HCS-B system is typically composed by a motorized Xuorescence microscope, a CCD camera that captures the images, and a digitizing system that stores the images in a large-capacity computer, with each step controlled by software for image acquisition and analysis. When applied to high-troughput screening (HTS) cell-based assays this novel technical concept allows cell research at large scale using multiwell plates and analyzing in bulk millions of cells per experiment. These systems may examine up to one single well per second and combine both high-temporal and spatial resolution. HCS-B is very eYcient for complex analyses that involve combinations of diVerent cell types and
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concentrations of agonists and antagonists, varying times of exposure as well as the type and number of parameters studied on each condition. In addition, the possibility of maintaining stable conditions of temperature and CO2 allows live cell functional assays. The detection in individual cells provides additional information on the responses of heterogeneous subpopulations that may diVer in their developmental stage, cell cycle phase, transfection status or other parameters. However, individual cell analysis may require special measurements (neurite outgrowth, nuclear localization, etc.), that reduce the speed required to be qualiWed as both HTS and HCS (Abraham et al., 2004; Borchert et al., 2005). 2.5. Laser capture microdissection Meaningful proteomic and genomic analysis demand the preparation of homogeneous cell populations. Flow-cytometry cell sorting may be used to purify a particular cell type in suspension but not easily from solid tissue samples. To improve cell isolation, several microdissection methods have been developed, but are often time-consuming and imprecise. LCM has been developed to automate and standardize microdissection, increasing reproducibility and accuracy of selecting targeted cells from a complex tissue for subsequent molecular analysis. LCM systems consist of an inverted microscope Wtted with a low-power near-infrared laser. Tissue sections are mounted on standard glass slides, and a transparent, 100-m thermoplastic Wlm is placed over the dry section. The laser energy melts Wlm in a precise location, binding it to the targeted cells, so that individual cells or a cluster of cells can be selected. After the appropriate cells have been selected, Wlm with adherent cells is removed, and the non-selected tissue remains in contact with the glass slide. Cells isolated by LCM can be extracted and submitted to a range of molecular analytical methods. Thus, mRNA measurements and cDNA microarrays of LCM-puriWed cells from microdissected tissues allow to compare loss of heterozygosity, detection of mutations and gene expression proWles between various cell types within a tissue. Mass spectrometric sequencing, peptide mass Wngerprinting, in-gel zymography, and Western blot have been used to identify proteins of interest. These approaches are particularly advantageous in identifying the diVerences between expression levels in normal, developing and diseased tissues (Curran et al., 2000; Zieziulewicz et al., 2003). 3. Cytomics in pharmacology and toxicology in vitro Cytomic analysis may be easily integrated among the essential strategies to study the interaction xenobiotic-cells for basic research, industrial development and the evaluation of therapeutic and toxic eVects of chemicals and biological compounds. In general, the aims of applying cytomics to pharmacology and toxicology can be summarized as follows (Fig. 1):
Fig. 1. Summary of the main processes susceptible of being analyzed by cytomic techniques in the investigation of the interactions between drugs/ toxicants and cells. The boxes represent major aspects of the interaction. In italics, particular phenomena that are frequently explored by cytomic techniques.
3.1. IdentiWcation and selection of speciWc cell subpopulations Cytomic assays allow to identify and eventually to purify a particular cell subpopulation of a complex cytome, by means of Xuorescent staining of surface markers, intracellular functional activities, morphological features or, more frequently, a combination of the above. As an example of this, by implementing a battery of assays we have recently demonstrated signiWcant functional diVerences among hepatoma cell subpopulations previously cloned by us using a Xow cytometry-based cell sorter on the basis of diVerential expression of P-glycoprotein in the parental hepatoma (O’Connor et al., 2005a). Lately, the multiparametric capacity of cytomic technologies is proven very useful for the identiWcation, characterization and isolation of stem cell populations, including embryonic, adult and tumoral stem cells. This is of special relevance, in view of the increasing interest in the cytotoxicity on stem cell populations in toxicological and pharmacological context (Hou et al., 2005). An essential requirement for FCM in this area is the identiWcation of the so-called side population (SP), a fraction enriched in totipotent stem cells in bone marrow (Goodell et al., 1996), human cancers including leukemia, solid tumors and primary cultures, and some adult normal tissues (Challen and Little, 2006). In these models, when cells are labelled with the membranepermeant DNA binding dye Hoechst 33342, a very small fraction of cells extrudes this dye via ABCG2/BCRP1 transporter and forms a dim tail extending from the normal cell populations, that allows to purify them via cell sorting. Technically, SP cells are deWned by the decreased Xuorescence emissions (red, FL7, and blue, FL6) of Hoechst 33342. The participation of the transporter in the eZux of Hoechst 3342 in SP cells is demonstrated by the disappearance of
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Fig. 2. An example of the sensitivity of Xow cytometry to detect and to characterize functionally a small subpopulation of relevant cells. (A) Detection of the side population (SP) of totipotent progenitor stem cells in mouse bone marrow. SP cells are deWned by the decreased Xuorescence emissions (red, FL7, and blue, FL6) of the vital DNA stain Hoechst 33342. (B) The participation of the ABCG2 transporter in Hoechst 33342 eZux in SP cells is demonstrated by the disappearance of SP cells following incubation with the eZux blocker verapamil. Plots represent a single experiment using the MoFlo cell sorter (Dako) and following the procedure described in Goodell et al. (1996).
SP cells following incubation with the eZux blocker verapamil (Fig. 2). 3.2. IdentiWcation of speciWc target cells or cells susceptible to drugs or xenobiotics The multiparametric capacity of cytomic methods allows to identify the expression of receptors speciWc for given drugs or the presence of structural and functional targets for a drug or xenobiotic within a cell population. In other cases, relevant parameters are related to the uptake, retention, biotransformation and eZux of xenobiotic compounds. From such parameters, the sensitivity of a particular cell type can be inferred. Most directly, cytomic assays can reveal on the whole cytomes or in speciWc subpopulations the eVects produced by the exposure to the drug or xenobiotic, thus providing evidence for cellular susceptibility or resistance (Lage et al., 2001; Herrera et al., 2003). Cytomic assays can be also applied to determine the sensitivity of eukaryotic or prokaryotic cells (Fig. 3) that have been submitted to genomic manipulation, which makes the cytomic approach an interesting tool to search for new models in cytotoxicity in vitro (Herrera et al., 2003). 3.3. Detection and quantiWcation of toxicity Cytomic techniques are widely used for quantitative and qualitative analysis of cell and organ toxicity (Alvarez-Barrientos et al., 2001). The main advantage of the cytomic approach derives from their multiparametric capacity (high-content assays), that provides multiple targets and endpoints to assess sublethal lesion and death in speciWc cell subpopulations. Thus, cytomic endpoints may represent early or late marker parameters along the cytotoxic process. On the other hand, the high velocity of many cytomic strategies allows the sequential analysis of large
numbers of cells per second, thus evidencing incipient or minoritary toxic eVects (Alvarez-Barrientos et al., 2001; Gómez-Lechón et al., 2003). 3.4. Characterization of drug and xenobiotic mechanisms Because of their multiparametric analytical power, their temporal (FCM) and topological (CLSM, HCS-B) resolution and the easy interaction with other “-omics”, cytomic strategies are frequently applied to explore the mechanisms of actions of drugs and toxics in human, animal and microbial cell models for biomedical (Gómez-Lechón et al., 2002), biotechnological (Perlman et al., 2004) and environmental studies (Lage et al., 2001). 3.5. Multiplexed analysis of soluble analytes A recent development of Xow cytometry, multiplexed assays consist in detecting separately but simultaneously soluble analytes (usually proteins or nucleic acid sequences) bound speciWcally by aYnity onto reactive Xuorescent microspheres of deWned optical properties. The design of such assays allows to quantify simultaneously several analytes in small volumes of sample. Because of this, multiplexed assays are advantageous over many conventional biochemical, immunological or molecular bulk standard techniques (Khan et al., 2004; Fuja et al., 2004). 3.6. High-troughput and high-content screening The development of current systems of ultrasensitive detection of Xuorescence, together with the fast rate of data acquisition provided by interfaced computing systems, allows the application of cytomic assays to robotized procedures of both high-content and high-troughput screening of compound libraries, based on cellular Xuorescence in
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Fig. 3. Correlation between genomic modiWcation of bacterial strains (A) and functional cytomic assays by means of end-point (B) or in Xuxo (C) Xow cytometric determination of intracellular oxidative stress. (A) OxyR, soxR and soxS are essential genes for antioxidant defence in Escherichia coli, and regulate other downstream genes by sensing hydrogen peroxide (oxyR) or superoxide (sodA, sodB). (B) E. coli B WP4 tester strains that have been made deWcient in oxyR, sodA and sodB genes exhibit increased levels of intracellular oxidative stress when analyzed by Xow cytometry using the superoxide-sensitive Xuorochrome hydroethidine, as described in Herrera et al. (2003). (C) OxyR mutants of E. coli B WP4 are more sensitive to hydroxyl radical than wild type controls when exposed to a source of hydroxyl radical (a combination of hydrogen peroxide plus copper sulphate), as shown by in Xuxo assay with the oxidant-sensitive Xuorogenic substrate dihydrorhodamine 123 (DHRh123). The increased rate of hydroxyl generation in the oxyR mutants over wild type cells is suggested by the enhanced production of Xuorescent rhodamine 123 from the parental leuko dye (DHRh123).
samples deposed in multiwell plates (Perlman et al., 2004; Smith and Giorgio, 2004).
use succinate or glucose as metabolic fuels (Juan et al., 1996).
3.7. The in Xuxo level of kinetic analysis by Xow cytometry
4. May cytomics provide in vitro alternative methods for toxicity assessment?
The dynamic nature of functional cytomic assays is exempliWed by a unique real-time kinetic approach, the in Xuxo level (O’Connor et al., 2005b), based on sequential examination of diVerent single cells from a population undergoing a dynamic process, that is triggered when cells of interest are being examined in the Xow cytometer (Fig. 3). Using the in Xuxo strategy, Xow cytometry has allowed us to focus on a highly speciWc biochemical process, the activity of the Na+/H+ transporter of plasma membrane (Dolz et al., 2004). In the other hand, an early application of the in Xuxo assay provided us a novel approach to calculate classical parameters of enzyme kinetics and to use them to reveal similarities and discrepancies between normal rat hepatocytes and rat hepatoma for the mitochondrial bioenergetic processes that
FCM is applied widely to the analysis of in vitro and ex vivo of many toxicity cellular markers albeit it never has been applied as a systematic and normalized unique strategy, in the form of an in vitro alternative method for chemical risk assessment. However, the recent developments in automatization and bioinformatics interfaced to FCM (Smith and Giorgio, 2004) and HCS-B instruments (Perlman et al., 2004) can be implemented into robotic procedures based on 96-well format assays in equipments available in growing number of laboratories. During the development of the European Contract “An evaluation of the reproducibility and transferability of Xow cytometric and confocal microscopic endpoints in an in vitro nephrotoxicity and in vitro metabolism models” we
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showed that a compact set of functional assays by Xow cytometry (the so-called “primary toxicity cytomic panel”, PTCP) reveals toxic eVects in models of acute, sustained and delayed exposure of tubular renal cells to nephrotoxins (cadmium chloride, cyclosporin an cis-platin) in sub-lethal concentrations (Alvarez-Barrientos et al., 2001). Our results suggest that cytomic functional assays may detect speciWcally early or transient changes in the process of cytotoxicity, which makes these assays advantageous over other tests limited to the quantiWcation of cell death as endpoint of cytotoxicity. In the second part of the mentioned research contract we showed how the application of PTCP allows to separate basal- and biotransformation-dependent cytotoxicity by means of the comparison of the cytotoxic eVects of two neuropharmacological substrates of cytochrome CYP2D6 (mianserin and imipramine) on the cell line V79 transfected stably with CYP2D6 and its control, mocktransfected cell line (Martínez-Romero et al., 2004). The Xow cytometric data on nephrotoxicity and metabolismdependent toxicity generated in this study were consistent with those obtained by confocal microscopy, another powerful cytomic technology, in an independent laboratory (Alvarez-Barrientos et al., 2001, 2003). Thus, for certain samples, FCM appears as a versatile tool that allows to approach diVerent levels of cellular complexity to reveal cytotoxic eVects and mechanisms in vitro or ex vivo, even in complex, heterogeneous cell populations.
Very recently, our laboratory has been enrolled in the EC sixth FP Integrated Project “A-Cute-Tox: Optimization and pre-validation of an in vitro test strategy for predicting acute human toxicity” (www.acutetox.org). This ambitious project (37 partners from 13 EC countries) is aimed to develop a simple and robust assay strategy to predict human acute systemic toxicity that could eventually replace current regulatory assays on whole animals. One of the speciWc objectives of A-Cute-Tox is to explore innovative tools and cell systems to identify new endpoints and strategies that anticipate better human and animal toxicity. Incorporation of cytomics to this project may deWne new endpoints of in vitro cytotoxicity to be incorporated into the predictive model or to provide new alerts and correctors of toxicity. In the Wrst part of this project we have been able of miniaturizing (96-well format) the cultures of rat and human cell lines, as well as of adjusting the experimental conditions for toxic treatment, cell resuspension, Xuorescent staining and Xow cytometric assay with the new Xow cytometers interfaced to 96-well plate sample loaders. This format of assay is comparable to that used for most of the current cellular assays of reference for in vitro cytotoxicity (Fig. 4). In this context, we have deWned the settings and calibration of the Xow cytometers for this type of assay and established the correlation in the rat hepatoma Fao between a Xow cytometric test of cell viability (propidium iodide exclusion)
Fig. 4. Analysis of digoxin cytotoxicity in three established cell lines by a Xow cytometric 96-well automated assay of propidium iodide exclusion. The established cell lines FAO (rat hepatoma), SH-SY5Y (human neuroblastoma) and A.704 (human kidney adenocarcinoma) were seeded in plastic 96-well plates and incubated in 10% FCS containing medium for 24 h (50% conXuence) in CO2 incubator at 37 °C. Then, the indicated concentrations of digoxin in 5% FCS containing medium were added and the plates kept for further 24 h. Then, cells were trypsinized and resuspended in their original wells in 5% FCS containing medium, in the presence of 5 g/mL propidium iodide (PI) and then analyzed automatically with the Cytomics FC500 MPL multiwellplate Xow cytometer (Beckman–Coulter). Data points represent triplicate wells from two separate experiments. The insert graph shows the Xow cytometric strategy to diVerentiate live cells (PI excluding cells) from apoptotic and necrotic by means of a plot correlating cell size (forward scatter) and PI uptake. Notice that neuroblastoma cells are much more sensitive to digoxin than the hepatoma or renal adenocarcinoma cells.
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and the Neutral Red uptake assay. For all the above considerations, we believe that both the technology (Xow cytometry) and the strategy (functional cytomic assays) may succeed in accomplishing meaningfully the proposed research project. Acknowledgements Sponsored by EC sixth FP projects A-Cute-Tox (LSHBCT-2004-512051) and Predictomics (LSHB-CT-2004504761), the Generalitat Valenciana (GV04B-143) and the Programa de Medicina Regenerativa de la Comunidad Valenciana. E.V. is a Bancaixa post-doctoral fellow. A.G. is recipient of a Leonardo fellowship. References Abraham, V.C., Taylor, D.L., Haskins, J.R., 2004. High content screening applied to large-scale cell biology. Trends in Biotechnology 22, 15–22. Abrous, D.N., Koehl, M., Le Moal, M., 2005. Adult neurogenesis: from precursors to network and physiology. Physiological Reviews 85, 523–569. Alvarez-Barrientos, A., O’Connor, J.E., Nieto-Castillo, R., MorenoMoreno, A.B., Prieto, P., 2001. Use of Xow cytometry and confocal microscopy techniques to investigate early CdCl2-induced nephrotoxicity in vitro. Toxicology In Vitro 15, 407–412. Alvarez-Barrientos, A., Callaghan, R.C., Huerta, E., Coecke, S., Martínez, A., Nieto, R., O’Connor, J.E., Prieto, P., Torralbo, P., 2003. Drugmetabolism dependent cytotoxicity of mianserin in vitro: application of a primary toxicity cytomic panel. In: VIII Congress of the Iberian Society of Cytometry, Madrid. Bernas, T., Grégori, G., Asem, E.K., Robinson, J.P., 2006. Integrating Cytomics and Proteomics. Molecular and Cellular Proteomics 5, 2–13. Borchert, K.M., Galvin, R.J., Frolik, C.A., Hale, L.V., Halladay, D.L., Gonyier, R.J., Trask, O.J., Nickischer, D.R., Houck, K.A., 2005. High-content screening assay for activators of the Wnt/Fzd pathway in primary human cells. Assay and Drug Development Technologies 3, 133–141. Challen, G.A., Little, M.H., 2006. A side order of stem cells: the SP phenotype. Stem Cells 24, 3–12. Curran, S., McKay, J.A., McLeod, H.L., Murray, H.I., 2000. Laser capture microscopy. Journal of Clinical Pathology: Molecular Pathology 53, 64–68. Dolz, M., O’Connor, J.E., Lequerica, J.L., 2004. Flow cytometric kinetic assay of the activity of Na+/H+ antiporter in mammalian cells. Cytometry 61A, 99–104. Eils, R., Athale, C., 2003. Computational imaging in cell biology. Journal of Cell Biology 161, 477–481. Figeys, D., 2004. Combining diVerent ‘omics’ technologies to map and validate protein–protein interactions in humans. BrieWngs in Functional Genomics and Proteomics 2, 357–365. Fuja, T., Hou, S., Bryant, P., 2004. A multiplex microsphere bead assay for comparative RNA expression analysis using Xow cytometry. Journal of Biotechnology 108, 193–205. Gómez-Lechón, M.J., O’Connor, J.E., Castell, J.V., Jover, R., 2002. Sensitive markers used to identify compounds that trigger apoptosis in cultured hepatocytes. Toxicological Sciences 65, 299–308. Gómez-Lechón, M.J., Ponsoda, X., O’Connor, J.E., Donato, T., Castell, J.V., Jover, R., 2003. Diclofenac induces apoptosis in hepatocytes by alteration of mitochondrial function and generation of ROS. Biochemical Pharmacology 66, 2155–2167. Goodell, M.A., Brose, K., Paradis, G., Conner, A.S., Mulligan, R.C., 1996. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. Journal of Experimental Medicine 183, 1797–1806.
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