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.     

Live-cell assay for detection of apoptosis by dual-laser flow cytometry using Hoechst 33342 and 7-amino-actinomycin D

This protocol describes a rapid and simple method for the identification of apoptotic cells. Owing to changes in membrane permeability, early apoptotic cells show an increased uptake of the vital DNA dye Hoechst 33342 (HO342) compared with live cells. The nonvital DNA dye 7-amino-actinomycin D (7-AAD) is added to distinguish late apoptotic or necrotic cells that have lost membrane integrity from early apoptotic cells that still have intact membranes as assayed by dye exclusion. The method is suitable to be combined with cell surface staining using Abs of interest labeled with fluorochromes that are compatible with HO342 and 7-AAD emissions. Surface antigen staining is carried out according to standard methods before staining for apoptosis. The basic assay can be completed in 30 min, and extra time is needed for cell surface antigen staining.     

Simultaneous cell cycle analysis and two-color surface immunofluorescence using 7-amino-actinomycin D and single laser excitation: applications to study of cell activation and the cell cycle of murine Ly-1 B cells

The DNA-binding, fluorescent dye 7-amino-actinomycin D (7AAD) is efficiently excited by the 488 nm laser line commonly used in flow cytometry, but yields fluorescence emission further into the red spectrum than alternative DNA-specific fluorochromes. In this report, we show that the spectral properties of 7AAD allow single-laser analysis of DNA content and cell cycle simultaneously with two cell surface markers labeled with fluorescein (green)-and phycoerythrin (orange)-conjugated antibodies. The use of 7AAD makes three-color analysis practical and feasible, using the most widely available flow cytometric instruments. The power of this technique was demonstrated in two systems. Staining of human peripheral blood lymphocytes (PBL) with 7AAD was demonstrated to be dependent on cell activation and chromatin conformation; PHA-stimulated cells which have become activated and express IL 2 receptors had greater 7AAD fluorescence than nonactivated, IL 2 receptor-negative cells. Cell cycle analysis of mouse splenocytes stained with fluorescent antibodies to IgM and to Ly-1 demonstrated that the proportion of S and G2 phase cells in native spleen varies strongly among the subsets of cells identified with these markers. Of particular interest was the striking finding that the Ly-1+/IgM+ subset (Ly-1 B cells) is greatly enriched for cells in the S phase fraction. This is important because Ly-1 B cells have been associated with the production of autoantibodies, and is consistent with reports that these cells have a lymphoblastoid or a plasmablast morphology. We hypothesize that Ly-1 B cells may belong to a subset of in vivo activated cells which are either rapidly proliferating or are arrested in S phase.     

Two- and three-color immunofluorescence using aminocoumarin, fluorescein, and phycoerythrin-labelled antibodies and single laser flow cytometry.

Antibodies coupled to 7-aminocoumarin (AMCA) emit a bright blue fluorescence under ultraviolet (UV) excitation and are therefore ideal for three-color immunofluorescence (IF) with fluorescein (FITC) and phycoerythrin (PE) labeled reagents; however, due to the different absorption spectra, the use of these fluorophores for multicolor flow-cytometric analysis requires a double light excitation source (e.g., two-laser system). We report a strategy which uses a single argon-ion laser to simultaneously excite AMCA, FITC, and PE, thus allowing the flow cytometric analysis of three immunological parameters. When the UV-visible argon-ion laser is fitted with an appropriate set of mirrors, the 35.1-363.8 nm (UV) and 488 nm wavelengths (accounting for 80 mW and 520 mW, respectively) are simultaneously generated; these lines can then be exactly focused on the same observation point by an achromatic cylindrical lens. A number of comparative analysis were performed with this instrumental set up to verify the sensitivity of AMCA IF and its possible application for multicolor immunophenotypic evaluation of blood cell subsets. When AMCA- and FITC-labeled antimouse Ig antibodies were assessed for their ability to detect limiting amounts of mouse monoclonal antibody bound to cells, the former was less sensitive than the latter. A number of factors, including differences in excitation energy (80 mW for AMCA and 520 mw for FITC) and extinction coefficients (1.9 x 10(4) for AMCA and 6 x 10(4) for FITC) could explain this result.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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.     

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.     

Correlating cell cycle with metabolism in single cells: combination of image and metabolic cytometry.

BACKGROUND: We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell. METHODS: A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence. RESULTS: Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G(1) phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G(2)/M cells. CONCLUSIONS: This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle.     

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.     

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.     

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.     

Detection of cell cycle subcompartments by flow cytometric estimation of DNA-RNA content in combination with dual-color immunofluorescence

BACKGROUND: Correlated flow cytometric measurements of phenotype and DNA-RNA content offer detailed information on cell cycle status of subpopulations in heterogeneous cell preparations in response to stimulation. We have developed a method for flow cytometric analysis of DNA-RNA content that has been optimized for simultaneous measurement of dual-color immunofluorescence. METHODS: Nucleic acid staining was performed at low pH in the presence of saponin. DNA was stained with 7-aminoactinomycin D (7-AAD) and RNA with pyronin Y(G) (PY); both dyes were used at low concentrations, and 7-AAD was exchanged with nonfluorescent actinomycin D after DNA staining to minimize fluorochrome-fluorochrome interactions. For cell surface antigen staining, allophycocyanin was combined with pH-independent Alexa488 instead of fluorescein-isothiocyanate (FITC) because FITC is pH sensitive. RESULTS: This method identified cell cycle subcompartments in CEM cells comparable to published results on cell lines using other dyes and staining methods. Measurement of DNA-RNA content in CD8 lymphocyte subsets of human peripheral blood mononuclear cells costimulated with CD3/CD28.2 showed that, after 48 h of stimulation, 80% of CD8(+) T cells were in the proliferative state, whereas 86% of CD8(+) non-T cells remained in G(0). CONCLUSIONS: This technique permits the clear identification of cellular subpopulations by phenotype and assessment of their cell cycle status.     

Phagocytosis of apoptotic cells assessed by flow cytometry using 7-Aminoactinomycin D

BACKGROUND: Apoptotic cells are recognized specifically by macrophages and are cleared rapidly by phagocytosis. However, the recognition mechanisms involved in the clearance of apoptotic cells by macrophages are still not fully understood. Therefore, new methods must be designed to better our understanding of the mechanisms of interaction between macrophages and apoptotic cells. 7-Aminoactinomycin D (7-AAD) is a fluorescent DNA-binding stain usually used as a single agent to detect apoptotic cells by flow cytometry. We propose the use of 7-AAD-stained apoptotic cells as targets for a new flow cytometry phagocytosis assay. METHODS: Murine T-cell lymphoma YAC-1 cells were treated with etoposide to induce apoptosis. Etoposide-treated YAC-1 target cells were stained subsequently with 7-AAD and then coincubated with resident peritoneal macrophages to allow phagocytosis. The samples were analyzed by flow cytometry. Macrophages that had phagocytosed 7-AAD-stained apoptotic cells were identified by their bright red fluorescence and the resulting values were expressed as the percentage of cells. RESULTS: The phagocytic cells appeared as a distinct population characterized by bright fluorescence, which could not be detected in the negative controls. The effects of a phagocytic enhancer (interferon-gamma [IFN-gamma]) or inhibitor (incubation at 4 degrees C) were assessed accurately with this flow cytometric method. CONCLUSIONS: We describe the use of 7-AAD in an assay that is easy and quick to perform. This flow cytometric-based assay allows the quantification of phagocytosis of apoptotic cells by macrophages.     

Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry.

Necrosis and apoptosis are two distinct modes of cell death which differ in morphology, mechanism and incidence. Membrane disruptants, respiratory poisons and hypoxia cause ATP depletion, metabolic collapse, cell swelling and rupture leading to inflammation. These are typical features of necrosis. Apoptosis plays a crucial role in embryogenesis and development and is also prevalent in tumours. It is characterised by cell shrinkage, chromatin condensation and systematic DNA cleavage. Apoptotic cells are rapidly engulfed by phagocytes, thus preventing inflammatory reaction to degradative cell contents. In vivo, apoptosis is almost impossible to quantify due to problems of heterogeneity and the short half-life of an apoptotic cell. In vitro, mechanistic studies are further complicated by a late phase of apoptosis where the cell membrane becomes permeable to vital dyes and which occurs in the absence of phagocytes. Here we describe a novel and rapid multiparameter flow cytometric assay which discriminates and quantifies viable, apoptotic and necrotic cells via measurement of forward and side light scatter (proportional to cell diameter and internal granularity, respectively) and the DNA-binding fluorophores Hoechst 33342 and propidium. It is anticipated that mechanistic studies of apoptosis in a variety of cell types will greatly benefit from this mode of analysis.     

Sensitive detection of RNAs in single cells by flow cytometry.

A rapid and sensitive fluorescent in situ hybridization method has been developed to probe RNA contents of individual cells by flow cytometry. Fixed cells in suspension were hybridized with 5' end-fluorophore-labeled oligodeoxynucleotides complementary to defined regions of the RNA of interest and analyzed by flow cytometry. With this method, we monitored combinations of histone H4 mRNA, 18S rRNA and 28S rRNA levels in synchronized HeLa S3 cells by multicolor analysis. A fluorescence signal equivalent to 1800 copies of histone H4 mRNA per cell was detected with signal-to-background ratio of 5.4. If non-specific binding of the fluorophore-labeled probe can be reduced, as few as 100 copies of mRNA of the size of H4 could be detected in individual cells by flow cytometry.     

Dynamics of single cell property distributions in Chinese hamster ovary cell cultures monitored and controlled with automated flow cytometry

Two important variables that are often not measured online in Chinese hamster ovary (CHO) cell cultures are cell number concentration and culture viability. We have developed an automated flow cytometry system that measured the cell number concentration, single cell viability based on propidium iodide (PI) exclusion, and single cell light scattering from bioreactor samples every 30 min. The bioreactor was monitored during batch growth, and then the cell number concentration was controlled at a set point during cytostat operation. NH₄Cl was added during steady state operation in cytostat mode to monitor the transient cell population response to adverse growth conditions. The automated measurements correlated well to cell concentration and viability determined manually using a hemacytometer. The described system provides a method to study mammalian cell culture physiology and dynamics in great detail. It presents a new method for the monitoring and control of animal cell culture.     

Dynamic assay of enzyme activities in single cells by flow cytometry.

Three enzymes in single cells were assayed dynamically by flow cytometry using four fluorogenic substrates. Acid phosphatase was determined with 7-bromo-3-hydroxy-2-naphtho-o-anisidine (naphthol AS-BI) phosphate and 4-methylumbelliferone (MU) phosphate, neutral esterase with fluorescein diacetate, and lactic dehydrogenase with NAD-sodium lactate. Fluorescence measurements obtained with the flow cytometer were converted into relative specific enzyme activities for single cells with molar fluorescence coefficients determined with a spectrofluorometer. Specific activities obtained from spectrofluorometric data were compared with activities calculated from flow cytometeric data. Flow cytometric assays gave lower specific single cell activities for 4-methylumbelliferone phosphate hydrolysis and for lactic dehydrogenase than did similar assays by standard spectrofluorometry. Product diffusion may be the greatest cause for this discrepancy.