Mixing small volumes for continuous high-throughput flow cytometry: performance of a mixing Y and peristaltic sample delivery

BACKGROUND: Online mixing for continuous high-throughput flow cytometry has not been previously described. A simple, general high-throughput method for mixing and delivery of submicroliter volumes in laminar flow at low Reynolds numbers would be widely useful. MATERIALS AND METHODS: We describe a micromixing approach that is compatible with commercial autosamplers, flow cytometry, and other detection schemes that require mixing of components that have been introduced into laminar flow. The scheme is based on a previous approach to high-throughput flow cytometry (HyperCyt, Kuckuck et al.: Cytometry 44:83-90, 2001). We showed that samples from multiwell 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, a particle sample flowing continuously is brought together in a Y with reagent samples from wells, which have been separated by bubbles. RESULTS: In the effluent stream, the particles and reagents are mixed, most likely as a result of peristaltic action, and reagents from individual wells can be resolved. The sample volumes that can be mixed with this technology are submicroliter in volume, and samples can be mixed at rates up to at least 100/samples per minute. With the current device, carryover between samples can be eliminated if the mixing system is flushed with several volumes of buffer. The anticipated throughput for screening is expected to be at least 20 samples per minute. CONCLUSIONS: The high-throughput approach and peristaltic mixing in HyperCytTM serve to integrate autosamplers with submicroliter detection volumes for analysis in flow cytometry or in microfluidic channels.     

High-throughput microfluidic mixing and multiparametric cell sorting for bioactive compound screening

HyperCyt, an automated sample handling system for flow cytometry that uses air bubbles to separate samples sequentially introduced from multiwell plates by an autosampler. In a previously documented HyperCyt configuration, air bubble separated compounds in one sample line and a continuous stream of cells in another are mixed in-line for serial flow cytometric cell response analysis. To expand capabilities for high-throughput bioactive compound screening, the authors investigated using this system configuration in combination with automated cell sorting. Peptide ligands were sampled from a 96-well plate, mixed in-line with fluo-4-loaded, formyl peptide receptor-transfected U937 cells, and screened at a rate of 3 peptide reactions per minute with approximately 10,000 cells analyzed per reaction. Cell Ca(2+) responses were detected to as little as 10(-11) M peptide with no detectable carryover between samples at up to 10(-7) M peptide. After expansion in culture, cells sort-purified from the 10% highest responders exhibited enhanced sensitivity and more sustained responses to peptide. Thus, a highly responsive cell subset was isolated under high-throughput mixing and sorting conditions in which response detection capability spanned a 1000-fold range of peptide concentration. With single-cell readout systems for protein expression libraries, this technology offers the promise of screening millions of discrete compound interactions per day.     

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.     

Cluster cytometry for high-capacity bioanalysis

Flow cytometry specializes in high-content measurements of cells and particles in suspension. Having long excelled in analytical throughput of single cells and particles, only recently with the advent of HyperCyt sampling technology, flow cytometry's multiexperiment throughput has begun to approach the point of practicality for efficiently analyzing hundreds-of-thousands of samples, the realm of high-throughput screening (HTS). To extend performance and automation compatibility, we built a HyperCyt-linked Cluster Cytometer platform, a network of flow cytometers for analyzing samples displayed in high-density, 1,536-well plate format. To assess the performance, we used cell- and microsphere-based HTS assays that had been well characterized in the previous studies. Experiments addressed important technical issues: challenges of small wells (assay volumes 10 μL or less, reagent mixing, cell and particle suspension), detecting and correcting for differences in performance of individual flow cytometers, and the ability to reanalyze a plate in the event of problems encountered during the primary analysis. Boosting sample throughput an additional fourfold, this platform is uniquely positioned to synergize with expanding suspension array and cell barcoding technologies in which as many as 100 experiments are performed in a single well or sample. As high-performance flow cytometers shrink in cost and size, cluster cytometry promises to become a practical, productive approach for HTS, and other large-scale investigations of biological complexity.     

High-throughput screening with HyperCyt flow cytometry to detect small molecule formylpeptide receptor ligands

High-throughput flow cytometry (HTFC), enabled by faster automated sample processing, represents a promising high- content approach for compound library screening. HyperCyt is a recently developed automated HTFC analysis system by which cell samples are rapidly aspirated from microplate wells and delivered to the flow cytometer. The formylpeptide receptor (FPR) family of G protein-coupled receptors contributes to the localization and activation of tissue-damaging leukocytes at sites of chronic inflammation. Here, the authors describe development and application of an HTFC screening approach to detect potential anti-inflammatory compounds that block ligand binding to FPR. Using a homogeneous no-wash assay, samples were routinely processed at 1.5 s/well (approximately 2500 cells analyzed/sample), allowing a 96-well plate to be processed in less than 2.5 min. Assay sensitivity and accuracy were validated by detection of a previously documented active compound with relatively low FPR affinity (sulfinpyrazone, inhibition constant [K(i)]=14 microM) from among a collection of 880 compounds in the Prestwick Chemical Library. The HyperCyt system was therefore demonstrated to be a robust, sensitive, and highly quantitative method with which to screen lead compound libraries in a 96-well format.     

An Automated High-Throughput Cell-Based Multiplexed Flow Cytometry Assay to Identify Novel Compounds to Target Candida albicans Virulence-Related Proteins

Although three major classes of systemic antifungal agents are clinically available, each is characterized by important limitations. Thus, there has been considerable ongoing effort to develop novel and repurposed agents for the therapy of invasive fungal infections. In an effort to address these needs, we developed a novel high-throughput, multiplexed screening method that utilizes small molecules to probe candidate drug targets in the opportunistic fungal pathogen Candida albicans. This method is amenable to high-throughput automated screening and is based upon detection of changes in GFP levels of individually tagged target proteins. We first selected four GFP-tagged membrane-bound proteins associated with virulence or antifungal drug resistance in C. albicans. We demonstrated proof-of-principle that modulation of fluorescence intensity can be used to assay the expression of specific GFP-tagged target proteins to inhibitors (and inducers), and this change is measurable within the HyperCyt automated flow cytometry sampling system. Next, we generated a multiplex of differentially color-coded C. albicans strains bearing C-terminal GFP-tags of each gene encoding candidate drug targets incubated in the presence of small molecules from the Prestwick Chemical Library in 384-well microtiter plate format. Following incubation, cells were sampled through the HyperCyt system and modulation of protein levels, as indicated by changes in GFP-levels of each strain, was used to identify compounds of interest. The hit rate for both inducers and inhibitors identified in the primary screen did not exceed 1% of the total number of compounds in the small-molecule library that was probed, as would be expected from a robust target-specific, high-throughput screening campaign. Secondary assays for virulence characteristics based on null mutant strains were then used to further validate specificity. In all, this study presents a method for the identification and verification of new antifungal drugs targeted to fungal virulence proteins using C. albicans as a model fungal pathogen.     

Sheath fluid control to permit stable flow in rapid mix flow cytometry.

BACKGROUND: Flow cytometry is a potentially powerful tool to analyze the kinetics of ligand binding, cell response and molecular assembly. The difficulty in adding reactant to cells, achieving adequate mixing, delivering those cells to the laser focal point and establishing stable flow, has historically limited flow cytometry to systems with reactions times longer than 5 s. With the advent of automated syringes and flow injection methods, sample injection times shorter than 1 s have become routine. However, an inherent problem in acquiring time courses starting under 1 s is that rapid sample introduction through the flow tip to the detection point perturbs laminar flow. The purpose of this work was to determine if stable flow could be reestablished more quickly if the sheath flow was reduced during sample introduction, returning to normal sheath and sample rates afterward. METHODS: We used programmable syringes and valves to control sample mixing as well as sheath and sample delivery through the flow tip to the detection point for stream-in-air detection. Stable flow was monitored by mean particle fluorescence during sample introduction. RESULTS: With no sheath reduction, stable flow recovered after more than 1 s. By reducing sheath flow during the short period (300 msec) of sample mixing and delivery, stable laminar flow recovered within 200 msec. CONCLUSIONS: This use of automated syringes to control both sheath and sample flow provides a potential for robust sample handling applicable to kinetic as well as high throughput flow cytometric analysis.     

High-throughput flow cytometry-based assay to identify apoptosis-inducing proteins

After sequencing the human genome, the challenge ahead is to systematically analyze the functions and disease relation of the proteins encoded. Here the authors describe the application of a flow cytometry-based high-throughput assay to screen for apoptosis-activating proteins in transiently transfected cells. The assay is based on the detection of activated caspase-3 with a specific antibody, in cells overexpressing proteins tagged C- or N-terminally with yellow fluorescent protein. Fluorescence intensities are measured using a flow cytometer integrated with a high-throughput autosampler. The applicability of this screen has been tested in a pilot screen with 200 proteins. The candidate proteins were all verified in an independent microscopy-based nuclear fragmentation assay, finally resulting in the identification of 6 apoptosis inducers.     

Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling

Flow cytometry allows high-content, multiparameter analysis of single cells, making it a promising tool for drug discovery and profiling of intracellular signaling. To add high-throughput capacity to flow cytometry, we developed a cell-based multiplexing technique called fluorescent cell barcoding (FCB). In FCB, each sample is labeled with a different signature, or barcode, of fluorescence intensity and emission wavelengths, and mixed with other samples before antibody staining and analysis by flow cytometry. Using three FCB fluorophores, we were able to barcode and combine entire 96-well plates, reducing antibody consumption 100-fold and acquisition time to 5-15 min per plate. Using FCB and phospho-specific flow cytometry, we screened a small-molecule library for inhibitors of T cell-receptor and cytokine signaling, simultaneously determining compound efficacy and selectivity. We also analyzed IFN-gamma signaling in multiple cell types from primary mouse splenocytes, revealing differences in sensitivity and kinetics between B cells, CD4+ and CD4- T cells and CD11b-hi cells.     

Uses and future applications of flow cytometry in immunotoxicity testing.

Flow cytometry is an emerging technology that has numerous applications to immunotoxicity testing. The use and development of high-speed single-cell laser-based assays capable of quantitation of fluorescence, light scatter, and electrical impedance measurements can provide important information on xenobiotic-induced toxicity in defined target cell populations. The purpose of this article is to briefly review established and emerging immunotoxicology assays that use flow cytometry. In the coming years it is likely that many new flow cytometry assays will be developed and validated that will improve the sensitivity and perhaps specificity of immunotoxicity testing. Since flow cytometry is readily adaptable to high-throughput screening, it is also likely that this technology will increasingly find its place in the preclinical testing of drugs and chemicals in the pharmaceutical and chemical industries.     

Photon-burst analysis in two-photon fluorescence excitation flow cytometry.

We studied the use of a dramatically reduced testing zone in combination with two-photon excitation and photon-burst analysis in high-throughput rare-event detection simulation using a modified flow cytometer. Two-photon excitation measurements were performed with a mode-locked titanium:sapphire laser. Fluorescence emission was measured with a photon-counting avalanche photodiode. Measured signal was analysed offline by autocorrelation and burst detection methods. Test samples were composed of full blood and orange fluorescent polystyrene nanospheres mixed in full blood. Results show that two-photon fluorescence excitation and time-correlation analysis provide a good signal-to-noise ratio for rare-event particle detection in a turbid sample environment.     

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.     

The proteomics of quiescent and nonquiescent cell differentiation in yeast stationary-phase cultures

As yeast cultures enter stationary phase in rich, glucose-based medium, differentiation of two major subpopulations of cells, termed quiescent and nonquiescent, is observed. Differences in mRNA abundance between exponentially growing and stationary-phase cultures and quiescent and nonquiescent cells are known, but little was known about protein abundance in these cells. To measure protein abundance in exponential and stationary-phase cultures, the yeast GFP-fusion library (4159 strains) was examined during exponential and stationary phases, using high-throughput flow cytometry (HyperCyt). Approximately 5% of proteins in the library showed twofold or greater changes in median fluorescence intensity (abundance) between the two conditions. We examined 38 strains exhibiting two distinct fluorescence-intensity peaks in stationary phase and determined that the two fluorescence peaks distinguished quiescent and nonquiescent cells, the two major subpopulations of cells in stationary-phase cultures. GFP-fusion proteins in this group were more abundant in quiescent cells, and half were involved in mitochondrial function, consistent with the sixfold increase in respiration observed in quiescent cells and the relative absence of Cit1p:GFP in nonquiescent cells. Finally, examination of quiescent cell-specific GFP-fusion proteins revealed symmetry in protein accumulation in dividing quiescent and nonquiescent cells after glucose exhaustion, leading to a new model for the differentiation of these cells.     

Quantification of CD59- mutants in human-hamster hybrid (AL) cells by flow cytometry

Mutation assay is an important approach in evaluating the genotoxic risk of potentially harmful environmental chemicals. The human-hamster hybrid (A(L)) cell mutagenesis system, based on the complement/antibody-mediated cytotoxicity principle, has been used successfully to evaluate the mutagenic potential of a variety of environmental toxicants. The A(L) cells contain a standard set of CHO chromosomes and a single human chromosome 11, which expresses several cell surface proteins including CD59 encoded by the CD59 gene at 11p13.5. A modified mutation assay by flow cytometry was developed to determine the yield of CD59- mutants after either radiation or chemical treatment. After incubation with phycoerythrin-conjugated mouse monoclonal anti-CD59 antibody, the CD59- mutant yields were determined by quantifying the fluorescence of the cells using flow cytometry. This method is faster and eliminates the commonly encountered toxicity problems of the complements with the traditional complement/antibody assay. By comparing the mutant fractions of radiation or chemically treated A(L) cultures using the two methods, we show here that the flow cytometry assay is an excellent substitute in providing an efficient and highly sensitive method in mutant detection for the traditional complement/antibody assay.     

Fluorescence flow cytometry and immunology applied to cancer

Antibodies against cell-surface determinants present on limited types of normal cells, such as differentiation antigens, offer an approach to detecting and characterizing tumor cells that is well-suited to fluorescence flow cytometry. This paper reviews the technology involved and its application in the study of hematologic malignancies, especially in the detection and characterization of tumor cells in leukemias and lymphomas.     

The curvHDR method for gating flow cytometry samples

Background  

High-throughput flow cytometry experiments produce hundreds of large multivariate samples of cellular characteristics. These samples require specialized processing to obtain clinically meaningful measurements. A major component of this processing is a form of cell subsetting known as gating. Manual gating is time-consuming and subjective. Good automatic and semi-automatic gating algorithms are very beneficial to high-throughput flow cytometry.     

Fluorescence anisotropy: a method for early detection of Alzheimer beta-peptide (Abeta) aggregation.

Time-resolved anisotropy measurements (TRAMS) have been used to study the aggregation of the beta-amyloid (Abeta) peptide which is suspected of playing a central role in the pathogenesis of Alzheimer's Disease (AD). The experiments, which employ small quantities of fluorescently-labelled Abeta, in addition to the untagged peptide, have shown that the sensitive TRAMS technique detects the presence of preformed "seed" particles in freshly prepared solutions of Abeta. More importantly, as 100 microM solutions of Abeta containing tagged Abeta at a concentration level of either 0.5 or 1 microM are incubated, the TRAMS prove capable of detection of the peptide aggregation process through the appearance of a continuously increasing "residual anisotropy" within the time-resolved fluorescence data. The method detects Abeta aggregation in its earliest stages, well before complexation becomes apparent in more conventional methods such as the thioflavin T fluorescence assay. The TRAMS approach promises to provide a most attractive route for establishment of a high-throughput procedure for the early detection of the presence of amyloid aggregates in the screening of biological samples.     

Improved sensitivity for solid-support invasive cleavage reactions with flow cytometry analysis

A new configuration of the solid-support invasive cleavage reaction provides a small reaction-volume format for high-sensitivity discrimination of nucleic acid targets with single nucleotide differences. With target concentrations as low as 2 amol/assay, the solid-support invasive cleavage reaction clearly distinguishes single base mutations. Two oligonucleotides tethered to the solid support hybridize to the target nucleic acid, forming a tripartite substrate that can be recognized and cleaved by Cleavase, a structure-specific 5'-nuclease. Each cleavage event yields fluorescence signal on the surface. When microspheres serve as the solid-support surface, analysis by fluorometer imparts real-time information about change in the reaction signal over time. Flow cytometry provides an alternative detection technology that collects endpoint information about the reaction signal on individual microspheres. A reaction volume of 10 microL with as few as 3000 microspheres is sufficient to distinguish single nucleotide differences at target concentrations less than 200 fM. This sensitivity level is within the range required for analysis of SNPs in genomic DNA. In addition, the flow cytometry format has multiplexing potential, making the microsphere-based invasive cleavage assay attractive for high-throughput genomic applications.     

Classifying antibodies using flow cytometry data: class prediction and class discovery

Classifying monoclonal antibodies, based on the similarity of their binding to the proteins (antigens) on the surface of blood cells, is essential for progress in immunology, hematology and clinical medicine. The collaborative efforts of researchers from many countries have led to the classification of thousands of antibodies into 247 clusters of differentiation (CD). Classification is based on flow cytometry and biochemical data. In preliminary classifications of antibodies based on flow cytometry data, the object requiring classification (an antibody) is described by a set of random samples from unknown densities of fluorescence intensity. An individual sample is collected in the experiment, where a population of cells of a certain type is stained by the identical fluorescently marked replicates of the antibody of interest. Samples are collected for multiple cell types. The classification problems of interest include identifying new CDs (class discovery or unsupervised learning) and assigning new antibodies to the known CD clusters (class prediction or supervised learning). These problems have attracted limited attention from statisticians. We recommend a novel approach to the classification process in which a computer algorithm suggests to the analyst the subset of the "most appropriate" classifications of an antibody in class prediction problems or the "most similar" pairs/ groups of antibodies in class discovery problems. The suggested algorithm speeds up the analysis of a flow cytometry data by a factor 10-20. This allows the analyst to focus on the interpretation of the automatically suggested preliminary classification solutions and on planning the subsequent biochemical experiments.     

Sample flow switching techniques on microfluidic chips

This paper presents an experimental investigation into electrokinetically focused flow injection for bio-analytical applications. A novel microfluidic device for microfluidic sample handling is presented. The microfluidic chip is fabricated on glass substrates using conventional photolithographic and chemical etching processes and is bonded using a high-temperature fusion method. The proposed valve-less device is capable not only of directing a single sample flow to a specified output port, but also of driving multiple samples to separate outlet channels or even to a single outlet to facilitate sample mixing. The experimental results confirm that the sample flow can be electrokinetically pre-focused into a narrow stream and guided to the desired outlet port by means of a simple control voltage model. The microchip presented within this paper has considerable potential for use in a variety of applications, including high-throughput chemical analysis, cell fusion, fraction collection, sample mixing, and many other applications within the micro-total-analysis systems field.