Kinetics of virus adsorption to single cells using fluorescent membrane probes and multiparameter flow cytometry

Different genetic stains of avian RNA tumor virus (ATV) were labeled with the fluorescent membrane probe R-18 (rhodamine conjugated to a hydrocarbon chain) and cellular receptors for virus infection were analyzed on a rapid, single-cell basis by a multiparameter cell sorter. Chicken cells genetically susceptible to various R-18 ATV were found to adsorb much more virus, as measured by increased fluorescent binding, than did genetically resistant chicken cells. Virus binding to receptor sites could be saturated with increased concentrations of labeled virus. This binding could be altered by removal of the polycation, polybrene, indicating the important influence of electrostatic forces. Correlated time measurements of virus binding to single cells were taken with these fluorescence measurements allowing for a minute-to-minute study of the kinetics of viral adsorption to resistant and susceptible cells. The ratio of fluorescence (proportional to the number of virions bound per cell) to light scatter (proportional to cell surface area) on a cell-to-cell basis was analyzed to examine the heterogeneity in fluorescent virion bound per unit cell surface area within a given cell type. With these calculations, it was found that a large amount, but not all, of observed fluorescence heterogeneity merely reflects differences in cell surface areas. However, there are significant differences in viral receptor site densities within this supposedly homogeneous population of cells. This study represents a successful application of fluorescent membrane probes and flow cytometry to the study of cellular responses to viral infection at the single-cell level. Sine large numbers of cells can be examined rapidly, small subpopulations of live virally susceptible or resistant cells can be cloned by multiparameter cell sorting.     

Quantification of bacterial invasion into adherent cells by flow cytometry

Quantification of invasive, intracellular bacteria is critical in many areas of cellular microbiology and immunology. We describe a novel and fast approach to determine invasion of bacterial pathogens in adherent cell types such as epithelial cells or fibroblasts based on flow cytometry. Using the CEACAM-mediated uptake of Opa-expressing Neisseria gonorrhoeae as a well-characterized model of bacterial invasion, we demonstrate that the flow cytometry-based method yields results comparable to a standard antibiotic protection assay. Furthermore, the quantification of intracellular bacteria by the novel approach is not biased by intracellular killing of the microbes and correctly discriminates between cell-associated extracellular and bona fide intracellular bacteria. As flow cytometry-based quantification is also applicable to other pathogen-host interactions such as the integrin-mediated internalization of Staphylococcus aureus, this approach provides a fast and convenient alternative for the quantification of bacterial uptake and should be particularly useful in elucidating the molecular mechanisms of pathogen-triggered host cell invasion.     

Characterization of bacteria by multiparameter flow cytometry

An arc-lamp based flow cytometer was used to obtain high resolution measurements of the light scattering characteristics and DNA contents of eight different bacteria. Light scatter profiles of bacteria are a useful first step when flow cytometry is used to characterize organisms. Scanning and transmission electron microscopy of the bacterial samples demonstrate that the structural basis of the light scattering profiles is not always clear, i.e. some organisms appear to have anomalous light scattering characteristics. The use of a third measurement parameter, DNA content, allowed much better discrimination of the organisms. Flow cytometry shows great promise as a method for the rapid discrimination and identification of bacterial populations.     

A reliable preparation of mono-dispersed chromosome suspensions for flow cytometry

Summary A reproducible, simple and rapid protocol was developed to prepare mono-dispersed chromosome suspensions for flow cytometric analysis. The procedure basically employes: 1. twice rinsing of monolayer cultures to eliminate dead cells and cellular debris, 2. mild fixation of mitotic cells with 1% acetic acid, and 3. ultra sonic treatment to release single metaphase chromosomes. The procedure takes less than 30 min. Isolated chromosomes are storable over months at 4° C. Despite mild fixation many biochemical studies and experiments applied on such fixed chromosomes purified and enriched using electronic sorting are feasible: 1. gene and restriction mapping, 2. cloning of specific gene sequences, and 3. gene frequency analysis.     

Identification and isolation of subpopulations of pleural cells by multiparameter flow cytometry

Multiparameter flow cytometry was used to identify and sort subpopulations of cells from pleural cell populations harvested from the rat without employing special stains or fluorochrome-labeled monoclonal antibodies. Cell parameters measured included electronic volume, axial light loss, 90° light scatter, and blue autofluorescence. Various bivariate combinations of these parameters were used to distinctly resolve pleural macrophages, eosinophils, mast cells, and lymphocytes. These subpopulations were separately sorted viably according to their unique electrooptical phenotypic characteristics in>90% purity. Our multiparameter flow cytometric approach, accordingly, provides a means by which pleural cell subpopulations may be easily obtained for subsequent in vitro study. Moreover, the general strategy for identifying and isolating these subpopulations may be usefully extended to the identification and isolation of subpopulations of cells occurring in other complex cell mixtures.     

Identification and isolation of subpopulations of pleural cells by multiparameter flow cytometry

Multiparameter flow cytometry was used to identify and sort subpopulations of cells from pleural cell populations harvested from the rat without employing special stains or fluorochrome-labeled monoclonal antibodies. Cell parameters measured included electronic volume, axial light loss, 90 degrees light scatter, and blue autofluorescence. Various bivariate combinations of these parameters were used to distinctly resolve pleural macrophages, eosinophils, mast cells, and lymphocytes. These subpopulations were separately sorted viably according to their unique electrooptical phenotypic characteristics in greater than 90% purity. Our multiparameter flow cytometric approach, accordingly, provides a means by which pleural cell subpopulations may be easily obtained for subsequent in vitro study. Moreover, the general strategy for identifying and isolating these subpopulations may be usefully extended to the identification and isolation of subpopulations of cells occurring in other complex cell mixtures.     

A single laser method for subtraction of cell autofluorescence in flow cytometry

In flow cytometry cell autofluorescence often interferes with efforts to measure low levels of bound fluorescent antibody. We have developed a way to correct for autofluorescence on a cell-by-cell basis. This results in improved estimates of real staining and better separation of the fluorescence histograms of stained and non-stained cells. Using a single laser, two-color fluorescence measurement system and two-color compensation electronics, autofluorescence and one fluorescent reagent are measured (rather than two fluorescent reagents). With fluorescein-conjugated antibodies the signal in the 515 to 555 nm range (green fluorescence) includes both fluorescein emission and part of the cellular autofluorescence. In the cases we have investigated, autofluorescence collected at wavelengths above 580 nm ("red") is well correlated with the green autofluorescence of the cells. A fraction of this red fluorescence is subtracted from the green fluorescence to produce an adjusted fluorescein output on which unstained cells have zero average signal. Use of this method facilitates the selection of rare cells transfected with surface antigen genes. Culture conditions affect the level of autofluorescence and the balance between red and green autofluorescence. When applied with fluorescein-conjugated reagents, the technique is compatible with the use of propidium iodide for live/dead cell discrimination.     

Characterization of bacteria by multiparameter flow cytometry.

An arc-lamp based flow cytometer was used to obtain high resolution measurements of the light scattering characteristics and DNA contents of eight different bacteria. Light scatter profiles of bacteria are a useful first step when flow cytometry is used to characterize organisms. Scanning and transmission electron microscopy of the bacterial samples demonstrate that the structural basis of the light scattering profiles is not always clear, i.e. some organisms appear to have anomalous light scattering characteristics. The use of a third measurement parameter, DNA content, allowed much better discrimination of the organisms. Flow cytometry shows great promise as a method for the rapid discrimination and identification of bacterial populations.     

A novel assay for assessment of HIV-specific cytotoxicity by multiparameter flow cytometry.

BACKGROUND: Assessment of CD8(+) T-cell activity is of significant importance for the evaluation of cellular immune responses to viral infections, especially in HIV. We present a new assay for the assessment of HIV-specific cytotoxicity by multiparameter flow cytometry. METHODS: Target cells, pulsed with peptide pools (Gag or Nef), were stained with 5- (and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), cultured with specific or nonspecific effector cells, and finally stained with propidium iodide (PI). Determination of cytolysis is based on the enumeration of viable target cells (CFSE(hi)PI(-)) in the test sample (target and specific effector cells) as compared with that of the viable target cells in the control sample (target and nonspecific effector cells). The (51)Cr-release assay and IFN-gamma ELISpot were performed by standard procedures. RESULTS: A comparison with the Cr-release showed that the two assays were strongly correlated (r = 0.67; P < 0.001) but the sensitivity of the flow cytometric assay was significantly higher (P < 0.05), and the reproducibility good (CV, 7.7%). Good correlation was also found with the ELISpot assay (r = 0.66; P < 0.01). CONCLUSION: This new assay provides both specific and sensitive results when employed for the detection of HIV-specific CTL and can be a valuable tool for the evaluation of cytolytic activity in vaccine trials or in HIV-infected subjects, especially if such responses are present at low levels.     

Tracking expression and subcellular localization of RNA and protein species using high-throughput single cell imaging flow cytometry

We report a high-throughput application of multispectral imaging flow cytometry (MIFC) for analyzing the expression and localization of both RNA and protein molecules in a heterogeneous population of cells. The approach was developed using polyadenylated nuclear (PAN) RNA, an abundant, noncoding RNA expressed by Kaposi's sarcoma-associated herpesvirus (KSHV) during the lytic phase of infection. High levels of PAN RNA are, in part, dependent on its interaction with poly(A)-binding protein C1 (PABPC1), which relocalizes from the cytoplasm to the nucleus of lytically infected cells. We quantitatively tracked the cytoplasmic to nuclear translocation of PABPC1 and examined how this translocation relates to the expression and localization of viral RNA and protein molecules in KSHV-infected cells. This high-throughput approach will be useful for other systems in which changes in subcellular localization of RNA and protein molecules need to be monitored simultaneously.     

Dual-laser flow cytometry of single mammalian cells.

An improved dual-laser flow cytometric system for quantitative analysis and sorting of mammalian cells has been developed using a low-power argon and high-power krypton laser as illumination sources, thus permitting the excitation of fluorescent dyes having absorption regions ranging from the ultraviolet to infrared. Cells stained in liquid suspension with fluorescent dyes enter a flow chamber where they intersect two spatially separated laser beams. Separate pairs of quartz beam-shaping optics focus each beam onto the cell stream. Electro-optical sensors measure fluorescence and light scatter signals from cells that are processed electronically and displayed as frequency distribution histograms. Cells also can be electronically separated and microscopically identified. The ease and versatility of operation designed into this system represent a marked technological improvement for dual-laser excited flow systems. Details of this instrument are described along with illustrative examples of cells stained with mithramycin and rhodamine and analyzed for DNA content, total protein, and nuclear and cytoplasmic diameter.     

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.     

Microscope-based multiparameter laser scanning cytometer yielding data comparable to flow cytometry data

We describe a computer-controlled 10 microns spot size laser scanning cytometer for making multiple wavelength fluorescence and scatter measurements of unconstrained cells on a surface such as a microscope slide. Designated areas of slides placed on a microscope stage are automatically scanned, and cells which generate above-threshold scatter or fluorescence values are found and individually processed to determine a list of measurement parameters. For each fluorescence or scatter measurement parameter, this list contains the integrated and peak values and bit pattern images of a scan window centered on the cell. The measurement time, the position of the cell on the slide, and two segmentation indices are also included in the list. Measurement time, cell position, and properties derived from the bit patterns are used interchangeably with integrated or peak measurement values as coordinates of multiproperty displays. Cells may be selected for counting, data display in various forms, or visual observation based on their meeting complex criteria among a chain of two property screens. Cells with selected properties may be viewed during an experiment or retrospectively. A designated specimen field may be repeatedly remeasured to perform kinetic cell studies. An argon ion and a HeNe- based laser instrument have been constructed and software has been written and evaluated with the specific goal of increasing the precision of propidium iodide-stained cellular DNA measurements. Some of the capabilities of the instrument and its current performance are described.     

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.     

Assessment of cell cycle-associated antigen expression using multiparameter flow cytometry and antibody-acridine orange sequential staining

A novel approach which enables direct assessment of the differential expression of cellular antigens in noncycling (G0) and cycling cell subpopulations is presented. The method involves flow cytometric analysis and sorting of cells stained by use of indirect immunofluorescence, followed by restaining using acid acridine orange, to relate the immunofluorescence of sorted lymphoid subpopulation(s) to cell proliferation status (i.e., G0 vs. G1 vs. S vs. G2 and M). In the present study, this technique successfully identifies the proliferation-associated modulation of a heterochromatin-associated antigen in pokeweed mitogen-stimulated human lymphoid cultures. The potential utility of this method for documenting early antigenic changes associated with the G0-G1 transition is discussed.     

A high-throughput cell-based toxicity analysis of drug metabolites using flow cytometry

The effects of liver enzymes on drug activities are important considerations in the drug discovery process. Frequently, liver microsomes are used to simulate first-pass metabolism in the liver; however, there are significant disadvantages to the microsome system. As an alternative, a simple cell-based, high-throughput system that allows for examination of metabolite activity is described. Using multiparameter flow cytometry and the low-volume, high-sample format of 96-well plates, it is possible to rapidly evaluate a dose-response curve for metabolites based on variables including initial compound concentrations, hepatocyte cell line metabolic activities, and time. Using HepG2 cells as a surrogate for hepatic metabolism of a potential therapeutic, the impact of metabolites on Jurkat cell death was measured by both propidium iodide dye exclusion and cell cycle analysis. While this system is not proposed to supplant liver microsome studies, this alternative assay provides a highly adaptable, low-cost, and high-throughput measure of drug metabolism.     

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.     

Single-cell analysis of siRNA-mediated gene silencing using multiparameter flow cytometry.

BACKGROUND: Use of synthetic short interfering RNAs (siRNAs) to study gene function has been limited by an inability to selectively analyze subsets of cells in complex populations, low and variable transfection efficiencies, and semiquantitative assays for measuring protein down-regulation. Intracellular flow cytometry can overcome these limitations by analyzing populations at the single-cell level in a high-throughput and quantitative fashion. Individual cells displaying a knockdown phenotype can be selectively interrogated for functional responses using multiparameter analysis. METHODS: Lck-specific siRNA was delivered into Jurkat T cells or peripheral blood mononuclear cells (PBMCs) to suppress endogenous Lck expression. Transfected cells were fluorescently stained for intracellular Lck and analyzed using multiparameter flow cytometry. The Lck(lo) Jurkat subpopulation was selectively analyzed for CD69 up-regulation and phospho-states of signaling proteins following T-cell receptor (TCR) stimulation. Surface expression levels of CD4 and CD8 on transfected CD3+ gated PBMCs were correlated with intracellular Lck levels. RESULTS: A subpopulation of Jurkat cells with reduced levels of Lck was clearly resolved from cells with wildtype levels of Lck. Both CD69 up-regulation and ZAP70 phosphorylation were suppressed in Lck(lo) cells when compared with those in Lck(hi) cells upon TCR stimulation. Knockdown of intracellular Lck in primary T lymphocytes reduced surface expression of CD4 in a dose-dependent manner. CONCLUSIONS: Multiparameter flow cytometry is a powerful technique for the quantitative analysis of siRNA-mediated protein knockdown in complex hard-to-transfect cell populations.