High resolution dual laser flow cytometry.

Flow cytometers based on optical sensing utilize external light sources and fluorescent dyes to measure one or more specific components or properties of individual cells or subcellular particles in liquid suspension. To provide for independent excitation of two dyes used in double staining experiments we have constructed a high resolution flow cytometer that uses two laser beams to provide two wavelengths of excitation. These beams are separated spatially so that cells flow through them sequentially, with a time separation of about 20 musec. Since the dyes are excited sequentially their emission occurs at different times and their emission spectra may overlap without causing any difficulty in analysis. We have developed new light collection optics that permit up to four measurements to be made on each cell. This approach greatly increases the number of dye combinations that can be used in flow cytometry, thus removing a significant limitation of single illumination instruments.     

Violet laser diodes in flow cytometry: an update.

INTRODUCTION: In previous studies we and others have demonstrated the usefulness of violet laser diodes (VLDs) as replacement laser sources for krypton-ion lasers on stream-in-air cytometers. Previously available VLDs had a maximum available power of less than 25 mW; this was sufficient for excitation of densely labeled cell surface antigens using fluorochromes such as Cascade Blue or Pacific Blue, but may have been insufficient for applications requiring higher levels of photon saturation, such as low-level expression of Cyan Fluorescent Protein (ECFP) in CFP-YFP FRET applications. In this follow-up study, we have tested more powerful VLDs emitting at 55 mW, and a beam-merged dual module VLD with 100 mW combined output, for their ability to excite a variety of violet-excited fluorochromes, including CFP. METHODS: A dual module VLD (two linear polarized VLDs with their beams merged by a polarized beam combiner) emitting at 404 nm was mounted on a BD FACSVantage DiVa stream-in-air cytometer. The individual polarized 55 mW beams or the 100 mW combined beams were used to analyze PBMCs labeled with the violet-excited probes Cascade Blue, Alexa Fluor 405, Cascade Yellow and Pacific Orange dyes. Violet-excited fluorescent microsphere mixtures with decreasing fluorescence levels were also used to detect the minimum sensitivity threshold and precision of these lasers. VLD excitation on a gel-coupled cuvette flow cytometer was used as a sensitivity baseline. RESULTS: The dual module 100 mW VLD gave both sensitivity and precision levels approaching that observed for lower-power sources on a cuvette cytometer. Single polarized VLD modules at 55 mW gave slightly decreased sensitivity for the microspheres standards and all the tested fluorochromes compared to the 100 mW source. CONCLUSIONS: While 55 mW laser sources performed adequately in the stream-in-air format, increasing the power to 100 mW did give a small but detectable increase in instrument sensitivity. This sensitivity level approached that of cuvette systems.     

Nine color eleven parameter immunophenotyping using three laser flow cytometry.

BACKGROUND: This study describes a three laser flow cytometer, reagents, and software used to simultaneously evaluate nine distinct fluorescent parameters on one cell sample. We compare the quality of data obtained with (1) full software compensation and (2) the use of partial spectral compensation of selected pairs of parameters in analog hardware, in combination with final software compensation. An application characterizing low frequency murine B cell subpopulations is given. METHODS: The fluorochromes used are: fluorescein (FITC), phycoerythrin (PE), Cy5PE and Cy7PE, excited at 488 nm by an argon laser; Texas Red (TR), allophycocyanin (APC), and Cy7APC excited at 595 nm by a pumped dye laser; and cascade blue (CB) and cascade yellow (CY) excited at 407 nm by a violet-enhanced krypton laser. Custom additions to commercial electronics and an extended optical bench allow the measurement of these nine parameters plus forward and side scatter light signals. RESULTS: We find the use of partial analog compensation reduces the variation in the background staining levels introduced by the compensation process. Novel B cell populations with frequencies below 1% are characterized. CONCLUSIONS: Nine color flow cytometry is capable of providing measurements with high information content. The choice of reagent-dye combinations and the ability to compensate in multi-parameter measurement space are crucial to obtaining satisfactory results.     

Analysis with flow cytometry of green fluorescent protein expression in leukemic cells.

BACKGROUND: The measurement of DNA content with propidium iodide (PI) in cells transfected with expression vectors encoding the green fluorescent protein (GFP) is a useful tool in studying a variety of biological functions of proteins within cells. The purpose of this study was to determine conditions of formaldehyde fixation that permit intracellular GFP fluorescence and adequate DNA histograms to be generated following transient transfection of cells with a GFP-encoding plasmid. Cell cycle analysis was also performed in GFP-positive cells. METHODS: The murine myeloid leukemic cell line, 32Dcl3, was used as the model system. Cells were transfected with a GFP-encoding plasmid (pEGFPC1). Following fixation in different formaldehyde concentrations and permeabilization with 70% ethanol, cells were stained with PI and analyzed by flow cytometry for GFP fluorescence and DNA content. Transfected cells were also analyzed for GFP fluorescence and DNA content following release from nocodazole block. RESULTS: Fixing cells in 0.51-1.75% formaldehyde concentrations prior to ethanol permeabilization resulted in 14-19% of transfected cells being GFP-positive, with acceptable coefficients of variation on the G(1) peak of DNA histograms. Analysis of cells synchronized to and released from the G(2)-M phase by nocodazole suggested that GFP-positive cells, when compared to GFP-negative cells, did not appear to progress out of G(2)-M following release from nocodazole block. Simultaneous detection of GFP fluorescence and DNA content by PI staining is possible following transient transfection of cells with a single expression vector encoding GFP. Our results demonstrate that GFP expression can be detected, using flow cytometry to perform cell cycle analysis in murine leukemic cells.     

Evaluation of platelet function by flow cytometry

Platelet function in whole blood can be comprehensively evaluated by flow cytometry. Flow cytometry can be used to measure platelet reactivity, circulating activated platelets, platelet-platelet aggregates, leukocyte-platelet aggregates, procoagulant platelet-derived microparticles, and calcium flux. Clinical applications of whole blood flow cytometric assays of platelet function in disease states (e.g., acute coronary syndromes, angioplasty, and stroke) may include identification of patients who would benefit from additional antiplatelet therapy and prediction of ischemic events. Circulating monocyte-platelet aggregates appear to be a more sensitive marker of in vivo platelet activation than circulating P-selectin-positive platelets. Flow cytometry can also be used in the following clinical settings: monitoring of GPIIb-IIIa antagonist therapy, diagnosis of inherited deficiencies of platelet surface glycoproteins, diagnosis of storage pool disease, diagnosis of heparin-induced thrombocytopenia, and measurement of the rate of thrombopoiesis.     

Evaluation of intranuclear BrdU detection procedures for use in multicolor flow cytometry.

BACKGROUND: Measurement of cell proliferation via BrdU incorporation in combination with multicolor cell surface staining would facilitate studies on cell subsets that require multiple markers for their identification. However, the extent to which the often harsh cell preparation procedures required affect the staining quality of more recently developed fluorescent dyes has not been assessed. METHODS: Three cell preparation protocols for BrdU measurement were compared for their ability to maintain fluorescent surface staining and scatter parameters of in vivo BrdU-labeled cells by flow cytometry. A 10-color fluorescent panel was developed to test the quality of surface staining, following cell treatment and the ability to perform BrdU measurements on even small B lymphocyte subsets. RESULTS: All cell preparation procedures affected the quality of fluorescent and/or scatter parameters to varying degrees. Paraformaldehyde/saponin-based procedures preserved sufficient fluorescent surface staining to determine BrdU incorporation rates among all splenic B cell subsets, including B-1a cells, which constitute roughly 0.5% of cells. Turnover rates of B-1a cells were similar to immature B cells and higher than those of the other mature B cell subsets. CONCLUSION: Paraformaldehyde/saponin-based cell preparation procedures facilitate detailed cell turnover studies on small cell subsets in vivo, revealing new functional information on rare cell populations.     

Analysis of UV-excited fluorochromes by flow cytometry using near-ultraviolet laser diodes.

INTRODUCTION: Violet laser diodes have become common and reliable laser sources for benchtop flow cytometers. While these lasers are very useful for a variety of violet and some ultraviolet-excited fluorochromes (e.g., DAPI), they do not efficiently excite most UV-stimulated probes. In this study, the next generation of InGaN near-UV laser diodes (NUVLDs) emitting in the 370-375-nm range have been evaluated as laser sources for cuvette-based flow cytometers. METHODS: Several NUVLDs, ranging in wavelength from 370 to 374 nm and in power level from 1.5 to 10 mW, were mounted on a BD Biosciences LSR II and evaluated for their ability to excite cells labeled with the UV DNA binding dye DAPI, several UV phenotyping fluorochromes (including Alexa Fluor 350, Marina Blue, and quantum dots), and the fluorescent calcium chelator indo-1. RESULTS: NUVLDs at the 8-10-mW power range gave detection sensitivity levels comparable to more powerful solid-state and ion laser sources, using low-fluorescence microsphere beads as measurement standards. NUVLDs at all tested power levels allowed extremely high-resolution DAPI cell cycle analysis, and sources in the 8-10-mW power range excited Alexa Fluor 350, Marina Blue, and a variety of quantum dots at virtually the same signal-to-noise ratios as more powerful UV sources. CONCLUSIONS: These evaluations indicate that near-UV laser diodes installed on a cuvette-based flow cytometer performed nearly as well as more powerful solid-state UV lasers on the same instrumentation, and comparably to more powerful ion lasers on a jet-in-air system, and. Despite their limited power, integration of these small and inexpensive lasers into benchtop flow cytometers should allow the use of flow cytometric applications requiring UV excitation on a wide variety of instruments.     

Evaluation of adenoviral vectors by flow cytometry

Biological assays for adenoviral gene therapy vectors have included conventional procedures initially developed to detect wild-type adenoviruses. Standard virological assays to quantitate adenoviruses rely on the virus to infect and replicate in the host cell until a cytopathic effect is observed. The appearance of plaques, colonies of rounded, enlarged cells containing infectious virions, usually takes 2 to 3 weeks to reach an endpoint. We describe a flow cytometric bioassay for adenovirus which shortens the time from when the infection takes place to the time that biological titer is determined. A fluorescent focus-forming assay was one of the first rapid adenoviral bioassays developed. Virus titer was determined using fluorescence immunocytochemistry to detect adenovirus proteins and microscopy to count fluorescent foci in cultures of adenovirus-infected cells. In this study, we describe a flow cytometric assay performed on cells stained for adenovirus hexon capsid protein, where virus titer is determined based on the dose-dependent appearance of hexon-positive cells. Adenovirus hexon detection in infected cells can provide data to determine virus titer, inducible promoter function in vector-complementing cells, and vector replication in complementation-deficient cells.     

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.     

Unique analytical capabilities of laser scanning cytometry (LSC) that complement flow cytometry

Flow cytometry has become an indispensable instrumentation in many disciplines of biology and medicine. There are some limitations of flow cytometry, inherent to the fact that the cells are measured in flow, which limit its usefulness in some applications. The microscope-based laser scanning cytometer (LSC) has many features similar to flow cytometry but few restrictions of the latter and therefore it is useful in many new applications. This review briefly outlines the applications that are unique to LSC, particularly related to its morphometric capabilities and the possibility of cell relocation. Potential future applications of LSC are also discussed.     

Performance analysis of a dual-buffer architecture for digital flow cytometry.

BACKGROUND: Most current commercial flow cytometers employ analog circuitry to provide feature values describing the pulse waveforms produced from suspended cells and particles. This restricts the type of features that can be extracted (typically pulse height, width, and integral) and consequently places a limit on classification performance. In previous work, we described a first-generation digital data acquisition and processing system that was used to demonstrate the classification advantages provided by the extraction of additional waveform features. An improved version of the system is discussed in this paper, focusing on dual-buffering to ensure increased pulse capture. A mathematical model of the system is also presented for performance analysis. METHODS: The second-generation system incorporates fast digitization of analog pulse waveforms, instantaneous pulse detection hardware, and a novel dual-buffering scheme. A mathematical model of the system was developed to theoretically compute the capture-rate performance. RESULTS: The capture rate of the system was theoretically analyzed and empirically measured. Under typical conditions, a capture rate of 8,000 pulses/s was experimentally achieved. CONCLUSIONS: Based on these results, the dual-buffer architecture shows great potential for use in flow cytometry.     

7-Amino-actinomycin D as a specific fluorophore for DNA content analysis by laser flow cytometry

A technique for DNA amount determination by flow cytometry based on the use of 7-amino-actinomycin D (7-amino-AMD), a fluorescent analogue of antibiotic actinomycin has been investigated, and a particular staining procedure has been developed. The procedure includes short fixation in 70% ethanol and staining for 20 min in 10(-5)M solution of 7-amino-AMD at pH7. The results of DNA content measurements are very reproducible. The histograms obtained have a coefficient of variation less than 3%. The absorption maximum of the complex of 7-amino-AMD with DNA is situated in the green spectrum region, making this stain particularly suitable for argon laser flow cytometry.     

Nonparametric flow cytometry analysis.

A nonparametric statistical test for the analysis of flow cytometry derived histograms is presented. The method involves smoothing and translocation of data, area normalization, channel by channel determination of the mean and S.D., and use of Bayes' theorem for unknown histogram classification. With this statistical method, different sets of histograms from numerous biological systems can be compared.     

Hydrodynamic orientation of sperm heads for flow cytometry.

Two specially shaped sample injection tubes orient flat-shaped mature sperm heads in a flow cytometer. Orientation allows control of a photometric difficulty experienced with some flow cytometers in the measurement of fluorescent and scattered light from sperm and other flat cells. Both relative DNA-stain content and a measure of cell flatness can now be determined simultaneously for each cell at the high rates possible with flow instruments.     

Simultaneous analysis of immunophenotype and apoptosis of murine thymocytes by single laser flow cytometry.

The study of the role of apoptosis in thymocyte development has been hampered by the lack of a means of directly immunophenotyping cells undergoing the early phase of apoptosis. This restriction has been overcome by single laser flow cytometry in which apoptosis is detected by Ethidium Bromide (EBr) staining and cell phenotype by binding of FITC-labelled antibody. The initial phase of apoptosis is observed as a cell population that stains faintly with EBr preceding the characteristically bright EBr-staining normally associated with cell death. Here we directly demonstrate using single laser flow cytometry that CD4+ CD8+ CD3low/CD3intermediate thymocytes undergo apoptosis in vitro in response to glucocorticoid treatment.     

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.