Data standards for flow cytometry

Flow cytometry (FCM) is an analytical tool widely used for cancer and HIV/AIDS research, and treatment, stem cell manipulation and detecting microorganisms in environmental samples. Current data standards do not capture the full scope of FCM experiments and there is a demand for software tools that can assist in the exploration and analysis of large FCM datasets. We are implementing a standardized approach to capturing, analyzing, and disseminating FCM data that will facilitate both more complex analyses and analysis of datasets that could not previously be efficiently studied. Initial work has focused on developing a community-based guideline for recording and reporting the details of FCM experiments. Open source software tools that implement this standard are being created, with an emphasis on facilitating reproducible and extensible data analyses. As well, tools for electronic collaboration will assist the integrated access and comprehension of experiments to empower users to collaborate on FCM analyses. This coordinated, joint development of bioinformatics standards and software tools for FCM data analysis has the potential to greatly facilitate both basic and clinical research--impacting a notably diverse range of medical and environmental research areas.     

Advances in flow cytometry for sperm sexing

This review presents the key technological developments that have been implemented in the 20 years since the first reports of successful measurement, sorting, insemination and live births using flow cytometry as a proven physical sperm separation technique. Since the first reports of sexed sperm, flow technology efforts have been largely focused on improving sample throughput by increasing the rate at which sperm are introduced to the sorter, and on improving measurement resolution, which has increased the proportion of cells that can be reliably measured and sorted. Today, routine high-purity sorting of X- or Y-chromosome-bearing sperm can be achieved at rates up to 8000 s⁻¹ for an input rate of 40,000 X- and Y- sperm s⁻¹. With current protocols, straws of sex-sorted sperm intended for use in artificial insemination contain approximately 2 × 10⁶ sperm. The sort rate of 8000 sperm s⁻¹ mentioned above corresponds to a production capacity of approximately 14 straws of each sex per hour per instrument.     

Quality assurance for polychromatic flow cytometry

This protocol outlines a three-part quality assurance program to optimize, calibrate and monitor flow cytometers used to measure cells labeled with five or more fluorochromes (a practice known as polychromatic flow cytometry). The initial steps of this program (system optimization) ensure that the instrument's lasers, mirrors and filters are optimally configured for the generation and transmission of multiple fluorescent signals. To determine the sensitivity and dynamic range of each fluorescence detector, the system is then calibrated by measuring fluorescence over a range of photomultiplier tube (PMT) voltages by determining the PMT voltage range and linearity (Steps 2-10) and validating the PMT voltage (Steps 11-17). Finally, to ensure consistent performance, we provide procedures to monitor the precision, accuracy and sensitivity of fluorescence measurements over time. All three aspects of this program should be performed upon installation, or whenever changes occur along the flow cytometer's optical path. However, only a few of these procedures need to be carried out on a routine basis.     

Real-time on-line flow cytometry for bioprocess monitoring

As the understanding of variation is the key to a good process and product quality one should pay attention to dynamics on the single-cell level. The basic idea of this approach was to qualify and quantify variations on the single-cell level during bioreactor cultivations by monitoring the expression of an eGFP tagged target protein (human membrane protein) using fully automated real-time, flow injection flow cytometry (FI-FCM). The FI-FCM system consists of a sampling- and defoaming- as well as of a dilution-section. It allows a very short monitoring interval (5 min) and is able to dilute the reactor sample by a factor ranging up to more than 10,000.In bioreactor cultivations of recombinant Pichia pastoris expressing the eGFP tagged target protein, high correlations (R² ≥ 0.97) between the FI-FCM fluorescent signal and other, however, population-averaged fluorescence signals (off-line fluorescence, in situ fluorescence probe) were obtained. FI-FCM is the only method able to distinguish between few cells with high fluorescence and many cells with low fluorescence intensity and proved that cells differ significantly from each other within the population during bioreactor cultivations. Single-cell fluorescence was distributed over a broad range within the cell population. These distributions strongly suggest that (a) the AOX-I promoter is leaky and (b) a fraction of the population is able to express more protein of interest within shorter time and (c) a fraction of the population does not express the fusion protein at all. These findings can help in the selection of high producing, stable strains. To show the platform-independency of the system, it has successfully been tested during bioreactor cultivations of three different strains (P. pastoris, Saccharomyces cerevisiae, Escherichia coli).Along with its applications in PAT, the FI-FCM could be used as a platform-independent (prokaryotes and eukaryotes) method in various other applications; for example in the closed-loop-control of bioprocesses using different kinds of fluorescent reporters, (waste- and drinking-) water analysis, clone selection in combination with FACS or even for surgery applications.     

Aptamers: versatile probes for flow cytometry

Aptamers are nucleic acid oligomers with distinct conformational shapes that allow them to bind targets with high affinity and specificity. Aptamers are selected from a random oligonucleotide library by their capability to bind a certain molecular target. A variety of targets ranging from small molecules like amino acids to complex targets and whole cells have been used to select aptamers. These characteristics and the ability to create specific aptamers against virtually any cell type in a process termed “systematic evolution by exponential enrichment” make them interesting tools for flow cytometry. In this contribution, we review the application of aptamers as probes for flow cytometry, especially cell-phenotyping and detection of various cancer cell lines and virus-infected cells and pathogens. We also discuss the potential of aptamers combined with nanoparticles such as quantum dots for the generation of new multivalent detector molecules with enhanced affinity and sensitivity. With regard to recent advancements in aptamer selection and the decreasing costs for oligonucleotide synthesis, aptamers may rise as potent competitors for antibodies as molecular probes in flow cytometry.     

A fast cell sampler for flow cytometry

A simple device has been developed for delivering samples into a flow cytometer. Designed with economy, simplicity, and flexibility in mind, this device, having only one moving part, can be used for sample volumes as small as 20 microliter, for virtually any form of cell sample container, and for a wide range of cell concentrations. It consists essentially of a lever-operated disc valve that allows the cell sample to be loaded into a loop of tubing and then to be injected into the cytometer nozzle under pressure from a saline source. The sampler has lifted the maximum analytical throughput of a FACS II cell sorter to better than 120 samples per hour.     

Exploitation of flow cytometry for plant breeding

In this review, the different applications of flow cytometry in plant breeding are highlighted. Four main breeding related purposes can be distinguished for flow cytometry: (i) Characterisation of available plant material, including screening of possible parent plants for breeding programs as well as evaluation of population biodiversity; (ii) Offspring screening after interspecific, interploidy or aberrant crosses; (iii) Ploidy level determination after haploidization and polyploidization treatments and (iv) Particle sorting, that allows separation of plant cells based on morphological or fluorescent characteristics. An overview and discussion of these various applications indicates that flow cytometry is a relatively quick, cheap and reliable tool for many breeding related objectives.     

Performance of in-line microfluidic mixers in laminar flow for high-throughput flow cytometry

We describe a micromixing approach that is compatible with commercial autosamplers, flow cytometry, and other detection schemes that require the mixing of components that have been introduced into laminarflow. The scheme is based on high-throughput flow cytometry (HyperCyt) where samples from multi-well plates that have been picked up by an autosampler can be separated during delivery by the small air bubbles introduced during the transit of the autosampler probe from well to well. Here, either cell or particle samplesflowing continuously and driven by a syringe are brought together in a Y with reagent samples from wells driven by a peristaltic pump. The mixing is driven by a magnetic microstirrer contained within the sample line. The mixing is assessed using fluorescence of both cell calcium responses and bead-based fluorescence unquenching. In the analysis stream, the particles and reagents are mixed with eithera "wire" or "bar". The bar is more efficient than the wire, and the efficiency of either depends on the spinning action. The high-throughput approach and mixing in HyperCyt integrate autosamplers with submicroliter detection volumes for analysis in flow cytometry or in microfluidic channels.     

7-Aminoactinomycin D for apoptosis staining in flow cytometry

All species of the genus Rhodnius have a characteristic red coloration in their salivary glands due to the presence of heme proteins. Some of these secreted proteins, known as nitrophorins (NPs), are responsible for many of the antihemostatic activities of Rhodnius saliva such as anticoagulant and antihistamine. Several NPs have been described (NP1-4 and NP7), where NP7 is the only one with affinity to phospholipid membranes. Computational prediction suggested that NP7 also has an extended N-terminal tail on signal peptide cleavage; however, the complementary DNA does not allow the determination of the exact site of signal peptidase cleavage. On the other hand, according to previous studies, the exact length of the N-terminus has important consequences for the nitric oxide binding properties of NP7. Here, a method was developed to select phospholipid membrane-attaching proteins from homogenized tissue for analysis by mass spectrometry. The method was used to determine the exact N-terminus of the ferriheme protein NP7 from homogenates of the salivary glands of 5th instar nymphal stages of Rhodnius prolixus.     

Cell labeling approaches for fluorescence-based in vivo flow cytometry

We provide an overview of the methods used to label circulating cells for fluorescence detection by in vivo flow cytometry. These methods are useful for cell tracking in small animals without the need to draw blood samples and are particularly useful for the detection of circulating cancer cells and quantification of circulating immune cells.     

Application of flow cytometry in chronobiology

A survey is given on flow cytometric techniques and their applications in chronobiology. Rapid automatic single cell measurements with a high rate (1,000-5,000 single cells/sec) enable the accumulation of large numbers of data at short intervals, thus obtaining important knowledge on circadian variations of cell proliferation in epidermis and other types of surface epithelium as well as hemopoiesis. In addition, the method has to some extent been used for monitoring cancer chemotherapy, and is thus available for the application in chronotherapy of malignancy. The introduction of rapid automatic techniques such as flow cytometry is a great advantage for any study on single cells and tissues where rhythmic variations in different functions have to be taken into consideration.     

Pattern recognition in flow cytometry.

BACKGROUND: Analytical flow cytometry (AFC), by quantifying sometimes more than 10 optical parameters on cells at rates of approximately 10(3) cells/s, rapidly generates vast quantities of multidimensional data, which provides a considerable challenge for data analysis. We review the application of multivariate data analysis and pattern recognition techniques to flow cytometry. METHODS: Approaches were divided into two broad types depending on whether the aim was identification or clustering. Multivariate statistical approaches, supervised artificial neural networks (ANNs), problems of overlapping character distributions, unbounded data sets, missing parameters, scaling up, and estimating proportions of different types of cells comprised the first category. Classic clustering methods, fuzzy clustering, and unsupervised ANNs comprised the second category.We demonstrate the state of the art by using AFC data on marine phytoplankton populations. RESULTS AND CONCLUSIONS: Information held within the large quantities of data generated by AFC was tractable using ANNs, but for field studies the problem of obtaining suitable training data needs to be resolved, and coping with an almost infinite number of cell categories needs further research.     

Uses of flow cytometry in hematology

Flow Cytometry, an analytical cytology technic which allows several parameters assessment in thousands cells per seconds is frequently used in hematology. The oldest application is the leucocyte count but several news applications have been developed for the analysis of cytomorphological and functional parameters of normal cells and for detection, classification of tumor cells. Most frequent applications are: The phenotyping of cells and the evaluation of relation between antigens and pathology. The cell cycle analysis which can be different in leukemic cells or which can be modified with chemotherapy or other agents. (Radiotherapy ...) Flow Karyotyping and in situ hybridization will extend applications of Flow Cytometry in hematology.     

A flow injection flow cytometry system for on-line monitoring of bioreactors

For direct and on-line study of the physiological states of cell cultures, a robust flow injection system has been designed and interfaced with flow cytometry (FI-FCM). The core of the flow injection system includes a microchamber designed for sample processing. The design of this microchamber allows not only an accurate on-line dilution but also on-line cell fixation, staining, and washing. The flow injection part of the system was tested by monitoring the optical density of a growing E.coli culture on-line using a spectrophotometer. The entire growth curve, from lag phase to stationary phase, was obtained with frequent sampling. The performance of the entire FI-FCM system is demonstrated in three applications. The first is the monitoring of green fluorescent protein fluorophore formation kinetics in E.coli by visualizing the fluorescence evolution after protein synthesis is inhibited. The data revealed a subpopulation of cells that do not become fluorescent. In addition, the data show that single-cell fluorescence is distributed over a wide range and that the fluorescent population contains cells that are capable of reaching significantly higher expression levels than that indicated by the population average. The second application is the detailed flow cytometric evaluation of the batch growth dynamics of E.coli expressing Gfp. The collected single-cell data visualize the batch growth phases and it is shown that a state of balanced growth is never reached by the culture. The third application is the determination of distribution of DNA content of a S. cerevisiae population by automatically staining cells using a DNA-specific stain. Reproducibility of the on-line staining reaction shows that the system is not restricted to measuring the native properties of cells; rather, a wider range of cellular components could be monitored after appropriate sample processing. The system is thus particularly useful because it operates automatically without direct operator supervision for extended time periods.