Reference standards for flow cytometry and application in comparative studies of nuclear DNA content

Nuclear DNA mass in cells from a reference species can be used to obtain high-resolution estimates of DNA mass from a target species. In our study of DNA mass in cells from 45 selected species, representing each of the major vertebrate classes, we have obtained values of from 1.5 to 110.0 pg of DNA. Because values in or near this range would be expected in the study of nuclear DNA mass in vertebrates and other organisms, the species in this report can provide a useful catalogue of references for comparative studies of DNA.     

Production of precise microbiology standards using flow cytometry and freeze drying.

BACKGROUND: Quality control standards provide a quantity of microorganisms for routine use in microbiology to demonstrate the efficacy of testing methods and culture media. Standards are normally prepared by diluting a culture of microorganisms to obtain a suspension that contains an estimated number of colony-forming units per milliliter. The variability and inaccuracy of these standards increase the potential for false results. Flow cytometry has been used extensively to prepare precise standards of Cryptosporidium and Giardia that contain exact numbers of organisms in a volume of liquid (1). However, the same levels of accuracy have yet to be obtained for bacterial quality control standards. METHODS: A modification of a Becton Dickinson FACScalibur flow cytometer enabled 30 bacterial cells to be sorted into a single droplet, mixed with a cryoprotective solution within the droplet, and frozen in liquid nitrogen. The frozen droplets were then freeze dried for stable preservation of the viable bacterial cells. RESULTS: A freeze-dried sphere 3 mm in diameter was produced, which contained 30 microorganisms. The within-batch variation for these freeze-dried spheres was no greater than two standard deviations, and the between-batch variation was less than one standard deviation. CONCLUSIONS: Bacterial reference controls can now be produced with consistent accuracy and unparalleled precision, thus enabling harmonization across the microbiological testing industry.     

Data reduction for spectral clustering to analyze high throughput flow cytometry data

Background  

Recent biological discoveries have shown that clustering large datasets is essential for better understanding biology in many areas. Spectral clustering in particular has proven to be a powerful tool amenable for many applications. However, it cannot be directly applied to large datasets due to time and memory limitations. To address this issue, we have modified spectral clustering by adding an information preserving sampling procedure and applying a post-processing stage. We call this entire algorithm SamSPECTRAL.     

Trout and salmon erythrocytes and human leukocytes as internal standards for ploidy control in flow cytometry

Control of technique and use of biological standards in flow cytometry have become increasingly important due to the wider use of the method for ploidy determination of malignant tumors in clinical research. Trout (TRBC) and salmon erythrocytes and human buffy coat leukocytes were selected for a study of factors influencing the DNA stainability. Whether standard and test cells were mixed before or after enzymatic treatment and staining was found to be critical for the ploidy comparisons. Otherwise, artifactual differences of at least 20% may be noted, leading to an overestimation of DNA aneuploidy. The time from staining to analysis had minimal effect, with some exceptions. The proportions of different cells in the sample had no influence, and nonlinearity of measurements was negligible. Diploid cells in normal endometrium and benign ovarian tumors, as well as the diploid fraction of aneuploid tumor cells, were systematically measured to have a DNA staining 5-7% above human leukocytes.     

Use of diploid and triploid trout erythrocytes as internal standards in flow cytometry.

DNA content determination requires the use of standards. Vindelov has shown the need to use two standards. Chicken and trout erythrocytes are commonly used, but they are not ideal standards. On the one hand, their DNA contents rarely frame the studied sample DNA content, and, on the other hand, as their base compositions are different in terms of A + T/G + C, their relative indices change according to the stains used. Use of triploid trout erythrocytes instead of chicken erythrocytes allows elimination of these two drawbacks; however, diploid trout must be differentiated from triploid trout. The present paper shows that an anatomic malformation is found with the triploid trout and so justifies the use of paired diploid and triploid trout as standards to measure nuclear DNA content.     

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.     

Fringe-scan flow cytometry

We describe the development of a scanning flow cytometer capable of measuring the distribution of fluorescent dye along objects with a spatial resolution of 0.7 micron. The heart of this instrument, called a fringe-scan flow cytometer, is an interference field (i.e., a series of intense planes of illumination) produced by the intersection of two laser beams. Fluorescence profiles (i.e., records showing the intensity of fluorescence measured at 20 ns intervals) are recorded during the passage of objects through the fringe field. The shape of the fringe field is determined by recording light scatter profiles as 0.25 micron diameter microspheres traverse the field. The distribution of the fluorescent dye along each object passing through the fringe field is estimated from the recorded fluorescence profile using Fourier deconvolution. We show that the distribution of fluorescent dye along microsphere doublets and along propidium iodide stained human chromosomes can be determined accurately using fringe-scan flow cytometry. The accuracy of fringe-scan shape analysis was determined by comparing fluorescence profiles estimated from fringe-scan profiles for microspheres and chromosomes with fluorescence profiles for the same objects measured using slit-scan flow cytometry.     

Photoacoustic flow cytometry

Conventional flow cytometry using scattering and fluorescent detection methods has been a fundamental tool of biological discoveries for many years. Invasive extraction of cells from a living organism, however, may lead to changes in cell properties and prevents the long-term study of cells in their native environment. Here, we summarize recent advances of new generation flow cytometry for in vivo noninvasive label-free or targeted detection of cells in blood, lymph, bone, cerebral and plant vasculatures using photoacoustic (PA) detection techniques, multispectral high-pulse-repetition-rate lasers, tunable ultrasharp (up to 0.8nm) rainbow plasmonic nanoprobes, positive and negative PA contrasts, in vivo magnetic enrichment, time-of-flight cell velocity measurement, PA spectral analysis, and integration of PA, photothermal (PT), fluorescent, and Raman methods. Unique applications of this tool are reviewed with a focus on ultrasensitive detection of normal blood cells at different functional states (e.g., apoptotic and necrotic) and rare abnormal cells including circulating tumor cells (CTCs), cancer stem cells, pathogens, clots, sickle cells as well as pharmokinetics of nanoparticles, dyes, microbubbles and drug nanocarriers. Using this tool we discovered that palpation, biopsy, or surgery can enhance CTC release from primary tumors, increasing the risk of metastasis. The novel fluctuation flow cytometry provided the opportunity for the dynamic study of blood rheology including red blood cell aggregation and clot formation in different medical conditions (e.g., blood disorders, cancer, or surgery). Theranostics, as a combination of PA diagnosis and PT nanobubble-amplified multiplex therapy, was used for eradication of CTCs, purging of infected blood, and thrombolysis of clots using PA guidance to control therapy efficiency. In vivo flow cytometry using a portable fiber-based devices can provide a breakthrough platform for early diagnosis of cancer, infection and cardiovascular disorders with a potential to inhibit, if not prevent, metastasis, sepsis, and strokes or heart attack by well-timed personalized therapy.     

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.     

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.     

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.     

High throughput flow cytometry

BACKGROUND: Conventional flow cytometry does not allow the rapid analysis of multiple samples. This has limited its uses in drug discovery, for which the standard for throughput is 100,000 samples per day. METHODS: We describe a simple method in which commercial peristaltic tubing is connected from a commercial autosampler to a flow cytometer. The samples are delivered via a peristaltic pump from source wells in a multiwell plate. The samples are separated by air bubbles. RESULTS: Throughput rates approach the limit of the autosampler (up to 100 wells per minute). Using optimal tubing and flow rates, particles remain within appropriate light scatter and fluorescence gates. The carryover between wells is typically less than 5% without and 1% with a wash step. The volumes of sample delivered are in the microliter scale. The approach has been validated with instruments from three manufacturers. CONCLUSIONS: Flow cytometry has potential throughput of 100,000 samples or more per day starting with the method described. The method is currently best suited to end-point assays. However, combined with high-speed sorting and single- cell assays, the number of assays could approach 1 billion per day.