Unraveling cell populations in tumors by single-cell mass cytometry

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High-content single-cell drug screening with phosphospecific flow cytometry

Drug screening is often limited to cell-free assays involving purified enzymes, but it is arguably best applied against systems that represent disease states or complex physiological cellular networks. Here, we describe a high-content, cell-based drug discovery platform based on phosphospecific flow cytometry, or phosphoflow, that enabled screening for inhibitors against multiple endogenous kinase signaling pathways in heterogeneous primary cell populations at the single-cell level. From a library of small-molecule natural products, we identified pathway-selective inhibitors of Jak-Stat and MAP kinase signaling. Dose-response experiments in primary cells confirmed pathway selectivity, but importantly also revealed differential inhibition of cell types and new druggability trends across multiple compounds. Lead compound selectivity was confirmed in vivo in mice. Phosphoflow therefore provides a unique platform that can be applied throughout the drug discovery process, from early compound screening to in vivo testing and clinical monitoring of drug efficacy.     

A single-cell assay of beta-galactosidase in recombinant Escherichia coli using flow cytometry

A flow cytometric method was developed for the assay of beta-galactosidase in single Escherichia coli cells. A new fluorogenic substrate for beta-galactosidase, C(12)FDG, contains a lipophilic group that allows the substrate to penetrate through cell membranes under normal conditions. When the substrate is hydrolyzed by intracellular beta-galactosidase, a green fluorescent product is formed and retained inside the cell. Consequently, the stained beta-galactosidase-positive cells exhibit fluorescence, which is detected by flow cytometry. This new assay was used to analyze the segregational instability caused by a reduction in specific growth rate of the plasmid-bearing cells in the T7 expression system. Induction results in a substantial accumulation of intracellular beta-galactosidase along with a rapid increase in the fraction of plasmid-free cells. Once the cells lose the plasmid, they no longer produce beta-galactosidase, which is reduced by at least half every generation; thus, after staining, the fluorescent, plasmid-bearing cells can be distinguished from the nonfluorescent, plasmid-free cells using flow cytometry. This article describes the feasibility of the flow cytometric assay for single E. coli cells and reports the optimal assay conditions. A direct relationship between beta-galactosidase activity and green fluorescence intensity was found, and the fractions of recombinant cells in batch cultures were analyzed after various levels of induction.     

A rapid method for evaluation of cell number and viability by flow cytometry

A simple, rapid and reliable method has been developed for assessing the number and viability of cells, as well as cell size, in suspension culture by the use of flow cytometry. Propidium iodide exclusion is used for viability determination and fluorescent beads serve as an internal standard for cell enumeration. The main advantages of this method are its ability to handle a large number of samples with a high degree of precision and its specificity in detecting viable cells quantitatively in a heterogeneous culture of living and dead cells and debris. The method shows only a fraction of the variation found in the haemacytometer/trypan blue counting method due to its very low operator dependence. CHO - Chinese hamster ovary; FCS - Foetal calf serum; FS - Forward scatter light; MTT - 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; NCS - newborn calf serum; PBS - Phosphate buffered saline; PI - Propidium iodide; SS - Side scatter light. This revised version was published online in July 2006 with corrections to the Cover Date.     

Microscopy-based multicolor tissue cytometry at the single-cell level.

Cytomics is a novel perspective from which to look at life. As with genomics and proteomics before, this discipline requires novel and innovative techniques and technologies to focus on its substrate of research--the cytome. With cytomics being the discipline that analyzes cellular systems and their interdependencies, advanced microscopy represents a key technology in cytomics research. Yet, conventional microscopy-based investigations, i.e., "look and conclude" analyses, do not meet the major cytomics criteria of 1) relating multiple parameters to each other, 2) within large populations of cells, 3) on a single-cell basis, and 4) in a quantitative and observer-independent manner. However, emerging improvements in the fields of fluorophore technology, sensitive fluorescence detection devices, and sophisticated image analysis procedures, are important and necessary steps into the cytomics era. Tissue represents an important class of cytomes, hence tissue cytometry--on the single cell level--can be expected to become an important cytomics technology. In this report, the techniques and technologies of microscopy-based multicolor tissue cytometry (MMTC) are outlined and applications are discussed, including the phenotypic characterization of tissue infiltrating leukocytes, in situ quantification of proliferation markers and tumor suppressors, and in situ quantification of apoptosis.     

Recent progress in mass spectrometry for single-cell metabolomics

Metabolites are the end products of cellular vital activities and can reflect the state of cellular to a certain extent. Rapid change of metabolites and the low abundance of signature metabolites cause difficulties in single-cell detection, which is a great challenge in single-cell metabolomics analysis. Mass spectrometry (MS) is a powerful tool that uniquely suited to detect intracellular small-molecule metabolites and has shown good application in single-cell metabolite analysis. In this mini-review, we describe three types of emerging technologies for MS-based single-cell metabolic analysis in recent years, including nano-ESI-MS based single-cell metabolomics analysis, high-throughput analysis via flow cytometry, and cellular metabolic imaging analysis. These techniques provide a large amount of single-cell metabolic data, allowing the potential of MS in single-cell metabolic analysis is gradually being explored and is of great importance in disease and life science research.     

Flow cytometry as an estimation tool for honey bee sperm viability

Flow cytometry is a method to conduct a multiparameter analysis of cells suspended in liquid and passing through a laser beam. Analyses of human and other mammal sperm using this method have already been performed but its application for insect semen is still the subject of investigation. Semen isolated from honey bee Apis mellifera seminal vesicles was dyed using SYBR-14 and propidium iodide (PI). The fluorescence of the SYBR-14 stained cells was analyzed in a green fluorescence channel (FL-1), while the PI fluorescence was analyzed in a red fluorescence channel (FL-3). Living and dead cell populations were separated using a density dot plot and the percentage of each in the sample was calculated. Flow cytometry seems to be an effective tool for assessing the viability of honey bee semen, solving the problems of distinguishing and counting the double-stained cells.     

Rapid assessment of bacterial viability by flow cytometry

The ability of a flow cytometer to rapidly assess microbial viability was investigated using three vital stains: rhodamine 123 (Rh123); 3,3'-dihexyloxacarbocyanine iodide [DiOC₆(3)] and fluorescein diacetate (FDA). Rh123 was found to clearly differentiate viable from non-viable bacteria. The methodology for staining bacteria with this dye was optimised. Rh123 was shown to stain and discriminate several different species of viable bacteria although this was not universal. Viable cells of Bacillus subtilis were found to stain better with FDAthan with Rh123. The results demonstrate the ability of flow cytometry to rapidly detect and estimate the viability of bacterial populations.Correspondence to: J. P. Diaper     

Hyperspectral cytometry at the single-cell level using a 32-channel photodetector

Despite recent progress in cell-analysis technology, rapid classification of cells remains a very difficult task. Among the techniques available, flow cytometry (FCM) is considered especially powerful, because it is able to perform multiparametric analyses of single biological particles at a high flow rate-up to several thousand particles per second. Moreover, FCM is nondestructive, and flow cytometric analysis can be performed on live cells. The current limit for simultaneously detectable fluorescence signals in FCM is around 8-15 depending upon the instrument. Obtaining multiparametric measurements is a very complex task, and the necessity for fluorescence spectral overlap compensation creates a number of additional difficulties to solve. Further, to obtain well-separated single spectral bands a very complex set of optical filters is required. This study describes the key components and principles involved in building a next-generation flow cytometer based on a 32-channel PMT array detector, a phase-volume holographic grating, and a fast electronic board. The system is capable of full-spectral data collection and spectral analysis at the single-cell level. As demonstrated using fluorescent microspheres and lymphocytes labeled with a cocktail of antibodies (CD45/FITC, CD4/PE, CD8/ECD, and CD3/Cy5), the presented technology is able to simultaneously collect 32 narrow bands of fluorescence from single particles flowing across the laser beam in <5 μs. These 32 discrete values provide a proxy of the full fluorescence emission spectrum for each single particle (cell). Advanced statistical analysis has then been performed to separate the various clusters of lymphocytes. The average spectrum computed for each cluster has been used to characterize the corresponding combination of antibodies, and thus identify the various lymphocytes subsets. The powerful data-collection capabilities of this flow cytometer open up significant opportunities for advanced analytical approaches, including spectral unmixing and unsupervised or supervised classification.     

Bacterial viability assessment by flow cytometry analysis in soil

Soil microhabitats and their heterogeneity are often considered to be among the most important factors affecting soil biotic communities. The microbial commu-nity has become one of the most important links in soil nutrient cycles and trophic components due to its role in biological processes, spatial and temporal dynamics, and physiological adaptation. Sandy-soil desert systems are characterized by fast water infiltration during the rainy season, high salinity, and low moisture availability in the upper soil layers. Plants have developed different ecophy-siological adaptations in order to cope with this harsh environment. The Tamarix aphylla is known to be one of the most commonly adapted plants, exhibiting a mechan-ism for secretion of excess salts as aggregates through its leaves. These leaves aggregate beneath the plant, creating 'islands of salinity'. Soil biotic components are, therefore, exposed to extreme abiotic stress conditions in this niche. The goal of this study was to examine the effect of T. aphylla on the live/dead bacterial population ratio on a spatial and temporal scale. The results emphasize the effect of abiotic factors, which changed on temporal as well as spatial scales, and also on the size of the active soil bacterial community, which fluctuated between 1.44% and 25.4% in summer and winter, respectively. The results of this study elucidate the importance of moisture availability and the 'island-of-salinity' effect on the active microbial community in a sandy desert system.     

Bacterial viability assessment by flow cytometry analysis in soil

Soil microhabitats and their heterogeneity are often considered to be among the most important factors affecting soil biotic communities. The microbial community has become one of the most important links in soil nutrient cycles and trophic components due to its role in biological processes, spatial and temporal dynamics, and physiological adaptation. Sandy-soil desert systems are characterized by fast water infiltration during the rainy season, high salinity, and low moisture availability in the upper soil layers. Plants have developed different ecophysiological adaptations in order to cope with this harsh environment. The Tamarix aphylla is known to be one of the most commonly adapted plants, exhibiting a mechanism for secretion of excess salts as aggregates through its leaves. These leaves aggregate beneath the plant, creating ‘islands of salinity’. Soil biotic components are, therefore, exposed to extreme abiotic stress conditions in this niche. The goal of this study was to examine the effect of T. aphylla on the live/dead bacterial population ratio on a spatial and temporal scale. The results emphasize the effect of abiotic factors, which changed on temporal as well as spatial scales, and also on the size of the active soil bacterial community, which fluctuated between 1.44% and 25.4% in summer and winter, respectively. The results of this study elucidate the importance of moisture availability and the ‘island-of-salinity’ effect on the active microbial community in a sandy desert system.     

Evaluation of a new reagent for covalent attachment of polyethylene glycol to proteins.

Methoxypolyethylene glycol of molecular weight 5000 was converted to a reactive succinimidyl carbonate form (SC-PEG). The usefulness of this new polymeric reagent for the covalent attachment of polyethylene glycol to proteins was evaluated. SC-PEG was found to be sufficiently reactive to produce extensively modified proteins under mild conditions within 30 min, showing the highest reactivity around pH 9.3. The commonly used succinimidyl succinate derivative of methoxypolyethylene glycol (SS-PEG) served as a reference standard to which the new reagent was compared. The stability of the polymer-protein linkages, studied on a series of PEG-modified bovine serum albumins, provided the single most important difference between the two activated polymers. Urethane-linked PEG-proteins obtained through the use of SC-PEG showed considerably higher chemical stability than SS-PEG-derived conjugates. The measured rate constants of aminolysis (using N alpha-acetyllysine) and hydrolysis showed that SC-PEG is slightly less reactive yet more selective of the two reagents. Hydrolysis of the active groups on SC-PEG was on average twofold slower than that on SS-PEG. The differences in the rates of aminolysis were even smaller than those in hydrolysis. PEG-trypsin conjugates produced by both activated polymers showed similar properties: they had no proteolytic activity, well-preserved esterolytic activity, and enhanced activity toward p-nitroanilide substrates. Michaelis-Menten constants of the modified enzymes were determined using N alpha-benzyloxycarbonyl-L-arginine p-nitroanilide. These measurements indicated that the attachment of PEG to trypsin caused an increase in both the rate of turnover of the substrate and its affinity toward the modified enzymes. Through a series of experiments involving the appropriate polymeric and low-molecular-weight model compounds, it was demonstrated that these increases in amidolytic activity were unrelated to tyrosyl residues acylation by either one of the activated polymers.     

Acid tolerance of Streptococcus macedonicus as assessed by flow cytometry and single-cell sorting

An in situ flow cytometric viability assay employing carboxyfluorescein diacetate and propidium iodide was used to identify Streptococcus macedonicus acid tolerance phenotypes. The logarithmic-phase acid tolerance response (L-ATR) was evident when cells were (i) left to autoacidify unbuffered medium, (ii) transiently exposed to nonlethal acidic pH, or (iii) systematically grown under suboptimal acidic conditions (acid habituation). Stationary-phase ATR was also detected; this phenotype was gradually degenerated while cells resided at this phase. Single-cell analysis of S. macedonicus during induction of L-ATR revealed heterogeneity in both the ability and the rate of tolerance acquisition within clonal populations. L-ATR was found to be partially dependent on de novo protein synthesis and compositional changes of the cell envelope. Interestingly, acid-habituated cells were interlaced in lengthier chains and exhibited an irregular pattern of active peptidoglycan biosynthesis sites when probed with BODIPY FL vancomycin. L-ATR caused cells to retain their membrane potential after lethal challenge, as judged by ratiometric analysis with oxonol [DiBAC(4)(3)]. Furthermore, F-ATPase was important during the induction of L-ATR, but in the case of a fully launched response, inhibition of F-ATPase affected acid resistance only partially. Activities of both F-ATPase and the glucose-specific phosphoenolpyruvate-dependent phosphotransferase system were increased after L-ATR induction, distinguishing S. macedonicus from oral streptococci. Finally, the in situ viability assessment was compared to medium-based recovery after single-cell sorting, revealing that the culturability of subpopulations with identical fluorescence characteristics is dependent on the treatments imposed to the cells prior to acid challenge.     

A new method for the rapid evaluation of gas vacuoles regeneration and viability of cyanobacteria by flow cytometry

Flow cytometric analysis for measuring gas vacuole regeneration and viability of cyanobacteria was developed. This novel approach distinguished between cyanobacteria with intact and collapsed gas vacuoles. By this method, sonicated cyanobacteria under illuminated conditions were shown to regenerate their gas vacuoles to the level of the untreated cells within 1–3 days. Ultrasonically treated cyanobacteria cultured under non-aerated and non-illuminated conditions did not regenerate their gas vacuoles. Combined with dual staining, viable and non-viable cyanobacteria are easily and rapidly quantified or enumerated by measuring the intensity of red fluorescence and green fluorescence. A high correlation was found between the numbers of viable and non-viable cyanobacteria with flow cytometric measurement.     

VEGF inhibitor (Iressa) arrests histone deacetylase expression: single-cell cotransfection imaging cytometry for multi-target-multi-drug analysis

Multi-target-multi-drug approaches are needed to accelerate the process of drug discovery screening and to design efficient therapeutic strategies against diseases that involve alterations in multiple cellular targets. Herein we report single-cell cotransfection imaging cytometry to quantitatively screen drug-induced off-target effects. Vascular endothelial growth factor (VEGF) and histone deacetylase (HDAC) genes amplified from the genomic DNA were cloned in fluorescently tagged gene constructs (RFP-HDAC/YFP-VEGF). These gene constructs were cotransfected in HEK-293 cells to explore the possibility of off-target effects of 4-phenylbutyrate and Iressa on the expression of VEGF and HDAC through single-cell imaging cytometry. Iressa (10 μM) treatment at the time of cotransfection or 48 h after cotransfection of RFP-HDAC/YFP-VEGF plasmids in HEK-293 cells resulted in off-target effects on HDAC expression. These results suggest possible applications of Iressa in the treatment of diseases in which expression of both HDAC and VEGF should be inhibited. 4-Phenylbutyrate (2.0 mM) did not show any off-target effects on VEGF expression. The developed quantitative multicolor live single-cell cotransfection imaging can be employed to select better drug combinations for faster screening and greater accuracy in multi-target-multi-drug analysis by increasing the on-target/desired off-target effects and eliminating the undesirable off-target effects.     

Flow cytometry for rapid assessment of viability after exposure to a quaternary ammonium compound

The use of flow cytometry to rapidly assess the viability of Pseudomonas spp. and Staphylococcus spp. after exposure to a quaternary ammonium compound (QAC) was investigated using rhodamine 123 (Rh 123), Stain A (LIVE Stain) accumulating in viable but not in dead cells (Live/Dead Bac light bacterial viability kit, Molecular Probes Inc., Eugene, OR, USA), and Sytox green (Molecular Probes) accumulating in dead but not viable cells. Staining conditions were optimized for each stain. The fraction of viable cells after exposure to benzalkonium chloride was determined by using the three staining techniques and colony counts on agar medium. For all Staphylococcus spp. tested there was a high correlation between the methods based on flow cytometry and colony counts irrespective of which stain was used. Although viable, all Pseudomonas spp. tested accumulated Rh 123 poorly and about 30% failed to accumulate LIVE stain as well. However, the correlation between colony counts and Sytox green labelling of Pseudomonas spp. was high. Our results indicate that flow cytometry together with live or dead cell labelling can be used to study the bactericidal effect of QACs. The methods based on LIVE stain and Sytox green were simpler and less time consuming than Rh 123 labelling. Only Sytox green could be used with all strains of Staphylococcvs and Pseudomonas tested.