John A. Steinkamp

A New Multiparameter Separator for Microscopic Particles and Biological Cells

A new flow‐system instrument for quantitative analysis and sorting of microscopic particles, particularly biological cells, based on multiple measurements of physical and biochemical properties has been developed. Cells stained with fluorescent dyes in liquid suspension enter a unique flow chamber where electrical and optical sensors measure cell volume, single‐ or two‐color fluorescence, and light scatter, and emerge in a liquid jet that is broken into uniform droplets. Sensor signals are electronically processed several ways for optimum cell discrimination and are displayed as pulse‐amplitude distributions using a pulse‐height analyzer. Processed signals trigger cell sorting according to preselected parametric criteria. Sorting is accomplished by electrically charging droplets containing the cells and electrostatically deflecting them into collection vessels. This instrument is described in detail with illustrative examples of experiments using polystyrene fluorescent microspheres, cultured human cells,...

Chapter 12 Methods and Applications of Flow Systems for Analysis and Sorting of Mammalian Cells1

Publisher Summary This chapter discusses the methods and applications of flow systems for analysis and sorting of mammalian cells. Flow systems offer several advantages when compared with the various static systems which analyze cells on slides. In most flow instruments, each cell is exposed to the light beam for only a few microseconds; thus, problems with fluorescence decay are minimized. The successful application of flow systems for rapid, single-cell analysis is critically dependent upon preparative techniques which maintain the cells in a monodispersed state during fixation, staining, and measurement. In instances where fluorescent staining techniques are employed, the quality and specificity of cellular staining must also be evaluated to ascertain the reliability of analytical results. Automated analytical systems that are designed to perform rapid and precise measurement of individual cells cannot be totally relied upon to distinguish fluorescent cellular debris or cell clumps from properly stained single cells. Therefore, sample preparation involving both the disaggregation of tissue into single-cell entities and cell staining plays an extremely important role in flow-system methodology.

Staining of mitochondrial membranes with 10‐nonyl acridine orange MitoFluor Green, and MitoTracker Green is affected by mitochondrial membrane potential altering drugs

BACKGROUND We set out to develop an assay for the simultaneous analysis of mitochondrial membrane potential and mass using the probes 10-nonyl acridine orange (NAO), MitoFluor Green (MFG), and MitoTracker Green (MTG) in HL60 cells. However, in experiments in which NAO and MFG were combined with orange emitting mitochondrial membrane potential (DeltaPsi(m)) probes, we found clear responses to DeltaPsi(m) altering drugs for both probes. METHODS The three probes were titrated to determine whether saturation played a role in the response to drugs. The effects of a variety of DeltaPsi(m) altering drugs were tested for MFG and MTG at probe concentrations of 20 nM and 200 nM and for NAO at 0.1 microM and 5 microM, using rhodamine 123 at 0.1 microM as a reference probe. RESULTS Incubation of GM130, HL60, and U937 cells with 2,3-butanedione monoxime (BDM), nigericin, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), 2,4-dinitrophenol (DNP), gramicidin, ouabain, and valinomycin resulted in increases of the fluorescence intensity for MFG or MTG with only a few exceptions. The fluorescence intensity of cells stained with 0.1 microM NAO increased following incubation with BDM, nigericin, and decreased for FCCP, CCCP, DNP, gramicidin, and valinomycin. The results with 5 microM NAO were similar. CONCLUSIONS MFG, MTG, and NAO appeared poor choices for the membrane potential independent analysis of mitochondrial membrane mass. Considering the molecular structure of these probes that favor accumulation in the mitochondrial membrane because of a positive charge, our results are not surprising. Cytometry 39:203-210, 2000. Published 2000 Wiley-Liss, Inc.

Analysis of fluorescence lifetime and quenching of FITC‐conjugated antibodies on cells by phase‐sensitive flow cytometry

Fluorescent antibodies are often used to measure the number of receptor sites on cells. The quantitative estimate of the number of receptor sites using this procedure assumes that the fluorescence intensity on a cell is proportional to the number of bound antibodies. Quenching may invalidate this assumption. For many fluorophores, intermolecular interactions and energy transfer between molecules in close proximity to one another results in self-quenching. This effect can occur in antibody probes with a high fluorochrome to protein (F/P) ratio. It can also occur due to close proximity antibodies relative to one another on a highly labeled cell surface. Since self-quenching is accompanied by a change in the fluorescence decay and a decrease in the fluorescence lifetime, it may be conveniently identified using fluorescence lifetime spectroscopy. In this paper we apply the phase-sensitive detection method to investigate the impact of self-quenching on fluorescence lifetimes by flow cytometry, using a model system consisting of FITC conjugated anti-mouse Thy1.2 antibodies bound to murine thymus cells. We show that in addition to the expected variation of lifetimes as a function of F/P ratio of the probes, the fluorescence lifetime diminishes also as a function of antibody labeling concentration on the cell surface. This is consistent with self-quenching effects expected at high densities of FITC molecules.

A new method for rapid and sensitive detection of bromodeoxyuridine in DNA-replicating cells☆

A new flow cytometric technique, involving differential fluorescence analysis of two DNA-binding fluorochromes, was used to quantify cellular incorporation of the base analog, bromodeoxyuridine (BrdU), into DNA over short time periods. During analysis of stained cells, the blue fluorescence signal of Hoechst 33342, which is quenched by BrdU-substituted DNA, was subtracted, on a cell by cell basis, from the green-yellow fluorescence signal of mithramycin, which remained stoichiometric to cellular DNA content. Bivariate contour profiles obtained for CHO cells pulse-labeled for 30 min showed that fluorescence quenching of Hoechst 33342 in BrdU-labeled, S phase cells produced fluorescence difference signals that were significantly greater than the difference signals from G1 and G2 + M phase cells. Analysis of L1210 cells demonstrated that the amount of BrdU detected was proportional to the length of the labeling period. The novel technique is simple, rapid, and mild; it produces minimal cell loss and does not significantly affect cellular moieties such as DNA, chromatin, or RNA.

[4] In Vitro and in Vivo measurement of phagocytosis by flow cytometry

Publisher Summary This chapter describes experimental approaches by which the phagocytic activity of virtually tens of thousands of cells in a population of macrophages can be rapidly measured in vitro on a cell-by-cell basis using flow cytometry. Phagocytosis measured by flow cytometry can be quantitatively described in two ways—namely, (1) the percentage of cells with engulfed particles can be determined and (2) individual particulate burdens within each cell can be expressed as frequency distributions showing the frequency of phagocytic cells with a given number of internalized particles. Cell populations should be maintained in a complete medium that is buffered with an organic buffer and not with sodium bicarbonate. Macrophages are exquisitely sensitive to a pH exceeding 7.4. The cells are analyzed for narrow angle light scatter (cell size) and for fluorescence (phagocytized spheres) as they flow through a flow cell and intersect a laser beam of exciting light. Optical sensors measure light scattering and fluorescence on a cell-by-cell basis and the signals are stored in the list mode data format in a computer. The data is then reprocessed and displayed as single- or two-parameter cell-size and fluorescence frequency distribution histograms.

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.

Flow cytometer for resolving signals from heterogeneous fluorescence emissions and quantifying lifetime in fluorochrome‐labeled cells/particles by phase‐sensitive detection

A phase‐sensitive flow cytometer has been developed that combines flow cytometry and fluorescence lifetime spectroscopy measurement principles to provide unique capabilities for making phase‐resolved measurements on fluorochrome‐labeled cells and particles. Stained cells are analyzed as they intersect a high‐frequency intensity‐modulated (sinusoid) laser excitation beam. Fluorescence is measured orthogonally using only a single‐channel optical detector. The detector output signals, which are phase shifted from a reference signal and amplitude demodulated, are processed by phase‐sensitive detection electronics to resolve signals from heterogeneous fluorescence emissions and quantify single‐component decay times. Results show signal phase shift and amplitude demodulation on fluorospheres; a detection limit threshold of 300–500 fluorescein molecules equivalence for excitation frequencies 1–30 MHz; a measurement precision (coefficient of variation) of 1.8% on alignment fluorospheres and 3.6% on cells stained ...

Flow cytometric characterization and classification of multiple dual‐color fluorescent microspheres using fluorescence lifetime

FlowMetrix (Luminex, Austin, TX) microspheres were recently introduced as a platform for bead-based assays involving antibodies, enzymes, toxins, and nucleic acids. The procedure involves classification of the microspheres by their orange and red fluorescence and quantitation of the BODIPY-tagged biological probes by their green fluorescence. In an attempt to increase the number of fluorochromes available for the biological probes, we explored the possibility of using excited singlet state lifetime as an alternative to one of the fluorochromes. For a set of 20 dual-color microspheres the excited singlet state lifetimes were measured using the total emissions (>515 nm), the orange emissions (515-600 nm), and the red emissions (>665 nm). The microspheres could not all be resolved in bivariates of fluorescence intensity versus excited singlet state lifetime. However, 13 of the microspheres could be resolved using the total emissions and lifetime. Although this result required both fluorochromes, the merits and limitations of this approach to other systems are briefly discussed.

Improved multilaser/multiparameter flow cytometer for analysis and sorting of cells and particles

An improved multilaser instrument has been developed for quantitative analysis and separation of biological cells and particles. Argon ion, krypton ion, and dye lasers are employed as excitation sources to sequentially illuminate cells labeled with multiple fluorochromes as they pass through an improved flow chamber that incorporates an electronic cell‐volume sensor and an optical measurement region. Detectors located on the axis of each excitation beam are used to measure axial light loss and forward light scatter. Multicolor fluorescence is measured using a five‐channel detector located orthogonal to the laser beam(s)‐cell stream intersection(s). Sequential measurements are made on a cell‐by‐cell basis to provide pulse height, area, and width signals that are made coincident in time by analog delay modules to increase data throughput. Analog electronics are used to compute real‐time ratios, sums, and differences of signals. Up to eight signals are acquired and displayed as single‐parameter frequency dis...