Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry

We provide a protocol for a high-resolution flow cytometry-based method for quantitative and qualitative analysis of individual nano-sized vesicles released by cells, as developed and previously described by our group. The method involves (i) bright fluorescent labeling of cell-derived vesicles and (ii) flow cytometric analysis of these vesicles using an optimized configuration of the commercially available BD Influx flow cytometer. The method allows the detection and analysis of fluorescent cell-derived vesicles of ～100 nm. Integrated information can be obtained regarding the light scattering, quantity, buoyant density and surface proteins of these nano-sized vesicles. This method can be applied in nanobiology to study basic aspects of cell-derived vesicles. Potential clinical applications include the detailed analysis of vesicle-based biomarkers in body fluids and quality control analysis of (biological) vesicles used as therapeutic agents. Isolation, fluorescent labeling and purification of vesicles can be done within 24 h. Flow cytometer setup, calibration and subsequent data acquisition can be done within 2-4 h by an experienced flow cytometer operator.     

Fluorescent inhibitors for the qualitative and quantitative analysis of lipolytic enzymes

We report on the determination of active enzyme components in pure and crude lipases, using fluorescent inhibitors for covalent modification and visualization of the enzymatically active proteins. Lipase-specific compounds are triacylglycerol analogs, namely 1,2(2, 3)-di-O-alkylglyceroalkylphosphonic acid-p-nitrophenyl esters, containing a fluorescent substituent bound to the omega-end of an alkyl chain. Inhibitors derived from single-chain alcohols, such as p-nitrophenyl esters of fluorescent alkyl phosphonates, react with lipases and esterases. The p-nitrophenyl ester bond is susceptible toward nucleophilic attack by the active serine of the lipolytic enzyme. This reaction is stoichiometric, specific, and irreversible. Stable lipid-protein complexes are formed which can be analyzed on the basis of their fluorescent signal. From fluorescence intensity the moles of active serine (enzyme) were accurately determined. A lipase-specific inhibitor was used for the analysis of a commercial lipase preparation from Rhizomucor miehei. After incubation of the enzyme with the fluorescent lipid, a single fluorescence band was observed after SDS-gel electrophoresis, indicating the presence of a single lipase in the crude enzyme material. A linear correlation was obtained between fluorescence intensity and the amount of enzyme. Using a combination of different inhibitors, we were able to discriminate between lipases and esterases.     

Detection of subgroups from flow cytometry measurements of heterotrophic bacterioplankton by image analysis.

BACKGROUND: Flow cytometry is an invaluable tool for the analysis of large series of samples in aquatic microbial ecology. However, analysis of the resulting data is often inefficient or does not reflect the complexity of natural communities. Because bacterioplankton assemblages frequently fall into several clusters with respect to their cellular properties, these subgroups seem to be a promising level of abstraction. Image analysis was used to detect clusters from flow cytometry data. The method was tested on a bacterial community under heavy protozoan grazing pressure. METHODS: A bivariate histogram of flow cytometry data was transformed into a gray-scale image for image analysis. After low-pass filtration, regional maxima were delimited by a watershed algorithm. The resulting areas were then used as gates on the original measurements. RESULTS: Three clusters could be detected from the bacterial assemblage. Protozoan grazing had a strong impact on the bacterial community, which could be analyzed in detail at the level of individual subgroups. CONCLUSIONS: Investigation at the level of bacterial subgroups allowed a more detailed analysis than whole-community statistics and delivered essential and ecologically meaningful information. Image analysis proved to be an adequate tool to detect the subgroups without a priori knowledge.     

Influence of sample size on image cytometry of DNA ploidy measurements

OBJECTIVE: To assess the number of nuclei required for significant image cytometry DNA ploidy measurements on one archival case of breast cancer. STUDY DESIGN: From one case of aneuploid DNA breast cancer, 18 subsets made up of 152-1,524 for the whole population of undamaged nuclei and made up of 74-735 epithelial nuclei had DNA measured. DNA ploidy type and five DNA ploidy indices, allowing DNA ploidy histogram interpretation were evaluated on each population. RESULTS: Three hundred nuclei were always sufficient for DNA typing, whereas reliable results for DNA ploidy indices required at least 750 nuclei. CONCLUSION: To DNA measure the above number of nuclei, fully automated image cytometry DNA ploidy measurements are required.     

A comparative study of quantitative stains for DNA in image cytometry.

In this study we examined the reproducibility of several stains used to measure nuclear DNA by image cytometry. The specimens were touch preparations of liver and testis from mouse and liver, intestine and brain from rat, fixed in either neutral formalin or Carnoy's solution. The tested stains included four Feulgen methods (pararosaniline, azure-A, thionin and acriflavine), the gallocyanine-chromalum stain and two fluorescent stains (acridine orange and propidium iodide). Absorbance measurements employed a video image analysis system; fluorescence measurements were from a scanning microspectrophotometer. The acriflavine-Feulgen stain was analyzed for both absorbance and fluorescence. All seven stains were quantitative for DNA and gave reproducible results. The absorbance measurements had a lower coefficient of variation (CV) than the fluorescence values. In a nested analysis of variance of the pararosaniline Feulgen stains, cell-to-cell variability accounted for 67% of the total variance; slide-to-slide, 9%; and batch-to-batch, 24%. These values did not change significantly when the staining was performed in an automatic staining machine. For DNA analysis using image cytometry, we conclude that the Feulgen staining technique is the most useful. In particular, acriflavine-Feulgen-stained cells fixed in Carnoy's fluid give the least variation between measurement values and the most accurate ratios between the separate ploidy groups. For fluorescence cytometry we recommend Carnoy's fixation and the acriflavine-Feulgen stain because of its narrow CV as compared to acridine orange and propidium iodide.     

Morphometry and cytometry correlated to quantitative autoradiography

Studies performed in various cell systems and designed to establish correlations between morphometric and functional parameters of individual cells are reviewed. Functional parameters were evaluated by utilizing quantitative 14C-autoradiography and measuring DNA, RNA and protein synthesis. Morphologic parameters were derived from the scanning of Feulgen-stained nuclei and the calculation of features related to shape, optical density and texture. A series of correlations between parameters of these two groups of features was established. Results such as the possibility of allocating cells to the G1, S and G2 phases by textural and densitometric features alone, without making use of the total DNA content and nuclear size, point to the power of this approach. The data, however, are not yet comprehensive enough to allow the interpretation of morphologic parameters in terms of cellular function on a large scale. It is emphasized that the measurement of the RNA synthesis rate will further promote the functional understanding of structural details and may help in making this approach useful for diagnostic purposes.     

Fluorescent biosensor for quantitative real-time measurements of inositol 1,4,5-trisphosphate in single living cells

The second messenger inositol 1,4,5-trisphosphate (IP(3)) plays a central role in the generation of a variety of spatiotemporally complex intracellular Ca(2+) signals involved in the regulation of many essential physiological processes. Here we describe the development of "LIBRA", a novel ratiometric fluorescent IP(3) biosensor that allows for the quantitative monitoring of intracellular IP(3) concentrations in single living cells in real time. LIBRA consists of the IP(3)-binding domain of the rat type 3 IP(3) receptor fused between the fluorescence resonance energy transfer pair cyan fluorescent protein and yellow fluorescent protein and preceded by a membrane-targeting signal. We show that the LIBRA fluorescent signal is highly selective for IP(3) and unaffected by concentrations of Ca(2+) and ATP in the physiological range. In addition, LIBRA can be calibrated in situ. We demonstrate the utility of LIBRA by monitoring the temporal relationship between the responses intracellular IP(3) and Ca(2+) concentrations in SH-SY5Y cells following acetylcholine stimulation.     

Preparation of monolayer smears from paraffin-embedded tissue for image cytometry

A method is described for the preparation of monolayer smears from paraffin-embedded tissue suitable for automated image analysis and DNA measurements. The proposed technique uses enzyme treatment and syringing for cell dispersal. Slide preparation is performed by centrifugal cytology. After Feulgen staining the quality of the monolayer smears is sufficiently high to enable visual morphologic evaluation. Automated DNA measurements using the Leyden television analysis system (LEYTAS) show coefficients of variation (CV) of 4.5% for the diploid cell population of the suspended tissue. This is approximately the same as the CV in fresh material from the same tumor. Formalin fixed trout red blood cells are used as reference cells. By applying image cytometry to paraffin-embedded tissue this method allows retrospective studies of, for instance, the significance of DNA content with regard to the behavior of a tumor.     

Clinical applications of image cytometry to human tumour analysis

Image cytometry (ICM) is widely applied to the automated screening, the detection, the diagnosis, the classification, the prognosis and the therapeutic follow-up of different types of cancers (breast, bladder, cervix,...). This review describes the analysis methods and the applications of nuclear image analysis, the determination of DNA content and the analysis of morphometry and of nuclear texture. DNA content analysis can contribute to a prognostic information in addition to other prognostic factors for breast, renal and prostate cancers. For ovarian cancer, aneuploidy seems to be related to prognosis. Bladder tumours with DNA aneuploidy were frequently of high malignancy while ploidy was significantly correlated to relapse risk. For digestive cancers, patients presenting DNA diploid tumours show a better survival than patients with aneuploid ones. Morphometry seems to be a more important criterion than other conventional prognostic factors of invasive breast and digestive carcinomas. A differential diagnosis between normal and neoplastic thyroids is more precise when based on a quantitative evaluation of texture associated to morphometry. Textural parameters permit the discrimination of two populations of patients having a different prognosis and could thus be an aid for prognosis in prostatic cancers. Morphonuclear parameters contribute to separate low and high grade bladder carcinomas. Although ICM was frequently reported, results from the reported examples were not always obvious. In conclusion, the measurements obtained with ICM could be helpful for a decision in several cancers but could not be a substitute for the classical approach of the pathologist.     

Fluorescent semiconductor nanocrystals in microscopy and flow cytometry

Quantum dots nanocrystals (Qdots or QDs), consisting of a CdSe core with a ZnS shell, are a novel class of fluorochromes with significant advantages over traditional organic fluorochromes and fluorescent proteins. QDs have a large extinction coefficient, high photostability, wide absorption and narrow emission spectra, and large Stokes shifts. These features make them desirable for both microscopy and flow cytometry. Applications of QD-conjugates with antibodies, streptavidin, and DNA or RNA probes have made it possible to produce highly stable multicolor specimens useful for scientific and diagnostic purposes. The current review describes the achievements in preparation of multicolor specimens based on QD-conjugates for microscopy and flow cytometry and outlines the requirements for microscope and flow cytometer reengineering for successful analysis of these specimens. However, despite considerable progress, two of the obstacles that preclude wider use of QDs include some of their chemical properties and the large size of QD-conjugates. Difficulties in the application of QDs are similar whether commercial or custom-made conjugates are used.     

Phosphorylation analysis by mass spectrometry: myths, facts, and the consequences for qualitative and quantitative measurements

The mass spectrometric analysis of protein phosphorylation is still far from being routine, and the outcomes thereof are often unsatisfying. Apart from the inherent problem of substoichiometric phosphorylation, three arguments as to why phosphorylation analysis is so problematic are often quoted, including (a) increased hydrophilicity of the phosphopeptide with a concomitant loss during the loading onto reversed-phase columns, (b) selective suppression of the ionization of phosphopeptides in the presence of unmodified peptides, and (c) lower ionization/detection efficiencies of phosphopeptides as compared with their unmodified cognates. Here we present the results of a study investigating the validity of these three arguments when using electrospray ionization mass spectrometry. We utilized a set of synthetic peptide/phosphopeptide pairs that were quantitated by amino acid analysis. Under the applied conditions none of the experiments performed supports the notions of (a) generally increased risks of losing phosphopeptides during the loading onto the reversed-phase column because of decreased retention and (b) the selective ionization suppression of phosphopeptides. The issue of ionization/detection efficiencies of phosphopeptides versus their unphosphorylated cognates proved to be less straightforward when using electrospray ionization because no evidence for decreased ionization/detection efficiencies for phosphopeptides could be found.     

Coincidence in high-speed flow cytometry: models and measurements

In flow cytometry, the coincident arrival of particles becomes a major problem when high sample rates are required. For the development of our high-speed photodamage flow cytometer (ZAPPER), it was of importance to understand the behavior of cells at flow rates of around 50,000-250,000 event/s. We developed and compared two models that describe the relation between the real cell rate and the detectable single cell rate. Both the Computer Simulation model and the Input/Output Device model show distinct optima for the cell rate. The models were compared to measurements performed on the ZAPPER-prototype. Fits of the two models to the experimental data were excellent for cycle times of 4 and 15 microseconds and acceptable for a 2 microseconds cycle time. A third model (Mercer WB, Rev. Sci. Instr. 37:1515-1521,1966) could be fitted to the experimental data, after the proportionality constant k was adapted to the experimental data. At a yield of detectable single cells of 70%, the maximum cell rates are 180,000, 100,000, and 40,000 cells/s for cycle times of 2, 4, and 15 microseconds, respectively. Based on these results we can now select an optimal cell rate for analysis and sorting based on criteria such as accepted cell loss. In addition, the advantages of reducing the cycle time can now be evaluated with respect to the costs of that modification.     

Single-cell mass cytometry adapted to measurements of the cell cycle

Mass cytometry is a recently introduced technology that utilizes transition element isotope-tagged antibodies for protein detection on a single-cell basis. By circumventing the limitations of emission spectral overlap associated with fluorochromes utilized in traditional flow cytometry, mass cytometry currently allows measurement of up to 40 parameters per cell. Recently, a comprehensive mass cytometry analysis was described for the hematopoietic differentiation program in human bone marrow from a healthy donor. The current study describes approaches to delineate cell cycle stages utilizing 5-iodo-2-deoxyuridine (IdU) to mark cells in S phase, simultaneously with antibodies against cyclin B1, cyclin A, and phosphorylated histone H3 (S28) that characterize the other cell cycle phases. Protocols were developed in which an antibody against phosphorylated retinoblastoma protein (Rb) at serines 807 and 811 was used to separate cells in G0 and G1 phases of the cell cycle. This mass cytometry method yielded cell cycle distributions of both normal and cancer cell populations that were equivalent to those obtained by traditional fluorescence cytometry techniques. We applied this to map the cell cycle phases of cells spanning the hematopoietic hierarchy in healthy human bone marrow as a prelude to later studies with cancers and other disorders of this lineage.     

Constructing quantitative models from qualitative mutant phenotypes: preferences in selecting sensory organ precursors

MOTIVATION: To study biology from the systems level, mathematical models that describe the time-evolution of the system offer useful insights. Quantitative information is required for constructing such models, but such information is rarely provided. RESULTS: We propose a scheme-based on random searches over a parameter space, according to criteria set by qualitative experimental observations-for inferring quantitative parameters from qualitative experimental results. We used five mutant constraints to construct genetic network models for sensory organ precursor formation in Drosophila development. Most of the models were capable of generating expression patterns for the gene Enhancer of split that were compatible with experimental observations for wild type and two Notch mutants. We further examined factors differentiating the neural fate among cells in a proneural cluster, and found two opposite driving forces that bias the choice between middle cells and the peripheral cells. Therefore, it is possible to build numerical models from mutant screening and to study mechanisms behind the complicated network.     

The psychological consequences of weight change trajectories: Evidence from quantitative and qualitative data

We use quantitative and qualitative data to explore the psychological impact of weight change among American adults. Using data from the Midlife Development in the United States (MIDUS) study, a survey of more than 3000 adults ages 25–74 in 1995, we contrast underweight, normal weight, overweight, obese I, and obese II/III persons along five psychosocial outcomes: positive mood, negative mood, perceived interpersonal discrimination, self-acceptance, and self-satisfaction. We further assess whether these relationships are contingent upon one's body mass index (BMI) at age 21. We find a strong inverse association between adult BMI and each of the five outcomes, reflecting the stigma associated with high body weight. However, overweight adults who were also overweight at age 21 are more likely than persons who were previously slender to say they were “very satisfied” with themselves. Results from 40 in-depth semi-structured interviews reveal similarly that persons who were persistently overweight or obese accept their weight as part of their identity, whereas those who experienced substantial weight increases (or decreases) struggle between two identities: the weight they actually are, and the weight that they believe exemplifies who they are. We discuss implications for stigma theory, and the ways that stigma exits and entries affect psychological well-being.     

C-value reassessment of plant standards: an image cytometry approach

Image cytometry (ICM) has been used to measure DNA 2C-values by evaluating the optical density of Feulgen-stained nuclei. This optical measurement is carried out using three basic tools: microscopy, digital video camera, and image analysis software. Because ICM has been applied to plants, some authors have remarked that studies should be performed before this technique can be accepted as an accurate method for determination of plant genome size. Based on this, the 2C-value of eight plants, which are widely used as standards in DNA quantifications, was reassessed in a cascade-like manner, from A. thaliana through R. sativus, S. lycopersicum, Glycine max, Z. mays, P. sativum, V. faba, to A. cepa. The mean 2C-values of all plants were statistically compared to the values reported by other authors using flow cytometry and/or ICM. These analyses demonstrated that ICM is an accurate and reliable method for 2C-value measurement, representing an attractive alternative to flow cytometry. Statistical comparison of the results also indicated Glycine max ‘Polanka’ as the most adequate primary standard. However, distinct authors have been advised that 2C DNA content of the reference standard should be close to that of the sample. As three further approaches also revisited the 2C-value of these eight plants, we have thus proposed a mean 2C-value for each eight species.