Simultaneous measurement of DNA content and cell-surface immunofluorescence of human bone marrow cells using a single laser flow cytometer.

This paper describes the measurement of S phase DNA content in human bone marrow subpopulations using a single laser method for bivariate analysis of DNA content and cell-surface immunofluorescence (s-IF). Low density (less than 1.077 g/ml) bone marrow cells were labeled with a panel of unconjugated monoclonal antibodies (MoAb) for the lymphoid (CD2 + CD19), T-lymphoid (CD2), B-lymphoid (CD19), erythroid (anti-glycophorin-A), myelomonocytic (CD13, CD33; single and as cocktail) and monocytic (CD14) lineages. A fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse label was used as second step. Unfixed, MoAb-labeled cells were incubated for 24 h with a hypotonic propidium iodide solution for DNA staining. Cells were analysed on a single-laser flow cytometer, operating at 488 nm. The effect of the combined staining protocol upon both s-IF and DNA stainability was evaluated. Only a slight decrease (mean: 29.0%) in s-IF intensity was observed after DNA staining. The percentages of immunofluorescent cells in the bone marrow samples of 10 normal individuals before and after DNA staining were essentially unchanged for all the MoAbs used. The DNA histograms of the immunophenotypically defined subpopulations were of excellent quality with a mean coefficient or variation of 1.8%. This procedure allows the assessment of very low levels of S-phase DNA content, as measured in normal low density blood cells of 8 healthy volunteers (mean 0.07%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Light scattering measurement in an arc lamp-based flow cytometer

The epi-illumination optics employed in most arc lamp-based flow cytometers may be modified so as to produce a dark-field configuration which facilitates highly sensitive detection of both forward and large angle light scattering in an instrument with a "jet on open surface" flow chamber. Forward scattering is detected at angles upwards from about 2 degrees, while large angle scattering includes angles above 18 degrees. Theoretical considerations suggest that large angle scattering measured around 20 degrees may be as efficient as that measured at 90 degrees for the purpose of distinguishing cells on the basis of intracellular structure. This was supported by the finding that dual parameter light scattering histograms of leukocyte suspensions obtained with the arc lamp-based instrument were closely similar to such histograms recorded with a laser-based instrument with the large angle detector at 90 degrees. Different species of bacteria could be distinguished by means of the dual parameter light scattering device, as could different species of sea algae. The sensitivity of the device is sufficient to measure 0.2 microns polystyrene particles in both forward and large angle scattering.     

Measurement in vitro of pulsatile arterial diameter using a helium-neon laser

A noncontacting in vitro measurement of pulsatile arterial diameter using a scanning optical micrometer is described. The major component of this system is a He-Ne laser whose beam scans the pulsating artery to be measured. The laser micrometer was integrated into a pulsatile perfusion apparatus that imposed various hemodynamic conditions on excised canine vessels. The laser system reliably tracked the pulsating arterial diameter at a particular longitudinal site as well as at various increments in the presence of an experimentally created stenosis. The He-Ne laser measured the radial motion of canine arteries and various vascular substitutes anastomosed in an end-to-end fashion. From these novel measurements, calculations were made of arterial compliance and bending stress, two biomechanical parameters that are implicated as potential causes of anastomotic intimal hyperplasia and graft failure. Although this device is inherently limited to in vitro use, it is a potentially useful instrument for vascular physiology and biophysics.     

Effect of low-power helium-neon laser irradiation on 13-week immobilized articular cartilage of rabbits

Influence of low-power (632.8 nm, Helium-Neon, 13 J/cm2, three times a week) laser on 13-week immobilized articular cartilage was examined with rabbits knee model. Number of chondrocytes and depth of articular cartilage of experimental group were significantly higher than those of sham irradiated group. Surface morphology of sham-irradiated group had rough prominences, fibrillation and lacunae but surface morphology of experimental group had more similarities to control group than to sham irradiated group. There were marked differences between ultrastructure features of control group and experimental group in comparison with sham irradiated group. Low-power Helium-Neon laser irradiation on 13-week immobilized knee joints of rabbits neutrilized adverse effects of immobilization on articular cartilage.     

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.     

A new principle of cell sorting by using selective electroporation in a modified flow cytometer

When a strong electric field pulse of a few microseconds is applied to biological cells, small pores are formed in the cell membranes; this process is called electroporation. At high field strengths and/or long pulse durations the membranes will be damaged permanently. This eventually leads to cell kill. We have developed a modified flow cytometer in which one can electroporate individual cells selected by optical analysis. The first experiments with this flow cytometer were designed to use it as a damaging sorter; we used electric pulses of 10 microseconds and resulting field strengths of 2.0 and 3.2 x 10(6) V/m to kill K562 cells and lymphocytes respectively. The hydrodynamically focused cells are first optically analyzed in the usual way in a square flow channel. At the end of this channel the cells are forced to flow through a small Coulter orifice, into a wider region. If optical analysis indicates that a cell is unwanted, the cell is killed by applying a strong electric field across the Coulter orifice. The wanted living cells can be subsequently separated from the dead cells and cell fragments by a method suitable for the particular application (e.g., centrifugation, cell growth, density gradient, etc.). The results of these first experiments demonstrate that by using very simple equipment, sorting by selective killing with electric fields is possible at rates of 1,000 cells/s with a purity of the sorted fraction of 99.9%.     

Setting up and running a microscope-based flow cytometer

A noncommercial flow cytometry system was set up in a laboratory with no engineering staff attached. The system is based upon a conventional fluorescence microscope and a new flow principle reported by H. Steen and T. Lindmo (Science 204:403, 1979). A few modifications and additions have been introduced. The instrument has proven to be a high-resolution system, easy to adjust and control, giving coefficients of variation of cellular DNA down to 0.8% and of fluorescent polyethylene microspheres down to 0.9%.     

A new approach for the analysis of testicular cells using a laser scanning cytometer.

The laser scanning cytometer (LSC) is a new laboratory tool that offers increased sensitivity and specificity compared to traditional technology. By combining the properties of the advantages of flow cytometry and immunohistochemistry, LSC-based analysis allows the automated evaluation of testicular cells in general and meiosis in particular. Testicular cell smears with previous staining by propidium iodide were analyzed by LSC. The results were compared with those for flow cytometry. LSC is a new, applicable methodology for analyzing spermatogenesis schedule.     

Display of three-parametric data acquired by a flow cytometer

A program for producing contour maps of three-parameter flow cytometric data is described. The program is written in Pascal and can be implemented on any microcomputer with a graphics screen.     

Simultaneous detection of NOS-3 protein expression and nitric oxide production using a flow cytometer

Nitric oxide (NO) is involved in the regulation of SMC proliferation during intimal hyperplasia as has been shown by the inhibitory effect on intimal hyperplasia of adenovirus-mediated ceNOS overexpression in injured arteries in pig. Good assays to quantify the NO-producing enzymes, i.e., NO synthases (NOS), are essential to analyze the mechanism of action of NO in this process. We have developed novel flow cytometric assays for the simultaneous detection of NOS-3 protein, using NOS-3 specific antibodies, and NO production using 4,5-diaminofluorescein-diacetate (DAF-2/DA). The presence of NOS-3 protein and NO production is demonstrated on human A549 and HepG2 cells infected with a NOS-3 adenovirus (Ad.NOS-3). A comparative study showed that the flow cytometric assays are equally sensitive as Western blot analysis, the citrulline assay, or the Sievers assay. On human endothelial and SMC, NOS-3 protein and NO production were simultaneously detected with the assays, both under basal conditions and after Ad.NOS-3transduction. Simultaneous analysis of NOS-3 protein and NO production, made possible by the here-described novel flow cytometric assays, is of significant value to those investigating NOS-3 and NO.     

Analysis of DNA-ploidy using laser scanning cytometer in brain tumors and its clinical application

There have been few reports on investigation of the cell kinetics of brain tumors using a laser scanning cytometer (LSC). We compared DNA analysis using a flow cytometer (FCM) with that by LSC using established cell lines of brain tumors, and we demonstrated the similarity of both analytical results. The DNA-index (DI) and proliferating index (PI) of various brain tumors were determined, and the results showed that these indices reflected the malignancy of the tumors. The DI values were higher for astrocytoma grade IV than for astrocytoma grade II, frequently showing an aneuploid pattern. The determination of DNA-ploidy using LSC, together with cryopathological diagnosis, was considered to be useful for deciding the area to be resected for glioma perioperatively and to be worth applying clinically.     

Integration of a barcode reader with a commercial flow cytometer.

This report describes the application and installation of a barcode reader on a standard EPICS Elite flow cytometer. The barcode reader system eliminates keyboard entry of sample information on the cytometer. The system automates the transfer of sample information already present in our laboratory database to the cytometer at run time. The system uses a standard "off-the-shelf" bar code wand with a personal computer keyboard interface and requires no additional software at run time. No typing of sample information is required by the operator at any stage of normal sample operation at the cytometer. All operations are automatically coded into the cytometry software using the macro functions of the software. Tubes are inserted into the tube reader and sample information is transferred automatically into the cytometer. We have found that the system allows rapid and continuous operation of routine clinical and research samples. This automated data entry also reduces the possibility of data input errors.     

Interfacing a flow cytometer computer with a PC-based patient information system

We have established an interface between our flow cytometer's computer and the personal computer (PC) which supports our patient database system. The PC has been equipped with a commercially available IEEE-488 bus interface board which is connected to the interface bus of the cytometer's Hewlett-Packard 9000/300 computer (HP). The PC is set as a bus device with the same address as that of the HP's printer. It is programmed to examine the stream of data sent to the printer and extract from it and store in an MS-DOS text file selected information which subsequently may be transferred to the database system.     

Characteristics of a simple, high-resolution flow cytometer based o a new flow configuration.

A method is described for the quantitative determination of free and bound solute concentrations in the cytoplasm of intact cells. The method includes (a) introduction of a gelatin gel reference phase (RP) into the cytoplasm; (b) diffusion of dissolved substances between cytoplasm and RP, (c) cell quenching to - 196 degrees C to prevent subsequent solute redistributions, (d) ultra-low temperature microdissection to isolate RP and cytoplasm samples, and (e) analysis of isolates for solute and water content. In normal oocytes of the salamander, Desmognathus ochrophaeus, free or RP Na+ and K+ are 21.0 +/- 1.1 and 128.8 +/- 2.4 mu eq/ml, respectively, and vary stoichiometrically in altered oocytes. Overall cytoplasmic concentrations are 75.2 +/- 2.7 mu eq Na+/ml and 88.6 +/- 1.5 mu eq K+/ml. Cytoplasmic chemical activities are 16.2 mu eq Na+/ml and 99.2 mu eq K+/ml, corresponding to activity coefficients of 0.22 and 1.12, respectively. The results demonstrate unambiguously that (a) oocytes actively transport Na+ and K+, and (b) cytoplasm has important binding properties which differentiate it from an ordinary aqueous solution. These cytoplasmic properties are investigated in the following paper.     

Calibration of a flow cytometer against a microphotometer for morphologic cell identification

The calibration of a flow cytometer against a microphotometer, to allow the correlation of cell morphology with fluorescence intensity, is described. Using three human lymphoblastoid cell lines, the photomultiplier amplification of the microphotometer and the flow cytometer that gave optimum linearity between fluorescence intensity and DNA content for the two instruments was established. Thereafter, at these settings, there was satisfactory linear agreement between the fluorescence intensity profiles, as measured by the flow cytometer and the microphotometer, of stained cell populations. Day-to-day variation was also minimal, and it was demonstrated that the application of this procedure can provide an alternative to the employment of the sorting facility of a flow cytometer for the morphologic identification of cell subpopulations during flow cytometric analysis.     

Increased immunofluorescence sensitivity using 532 nm laser excitation.

OBJECTIVE: We evaluated the use of a high power, diode pulsed solid-state laser emitting 532 nm light for immunofluorescence applications. We compared the sensitivity and utility of this laser with the standard 488 nm excitation. METHODS: A flow cytometer was equipped with both a 488 nm and a 532 nm laser; fluorescence emissions from each laser were collected using the same filters and the same detector system. Cells or compensation beads (e.g. latex beads coated with anti-kappa antibodies) were stained with monoclonal antibodies conjugated to phycoerythrin (PE) as well as the PE tandem dyes TRPE, Cy5PE, Cy5.5PE, and Cy7PE. The sensitivity of detection of these reagents as well as those in heavily compensated channels was quantified by measuring the spreading error for a primary detector into a secondary detector. RESULTS: Measurement of the fluorescence emission of PE and PE-tandem dyes was considerably more sensitive when using 532 nm excitation (150 mW) as compared with 488 nm excitation (20 mW). In addition, as the absolute number of photoelectrons collected was greater, there was less measurement-error-induced spread into the compensated channels. As an example, when comparing the spreading error of PE labeled cells into the TRPE detector, the green laser was found to be 15-fold more sensitive as compared with the blue laser. In addition, the blue laser produced more autofluoresent signal from cells as compared with the green laser. Together, these advantages of the 532 nm excitation line provides for a significantly improved detection of immunofluorescence staining.     

Extended application of flow microfluorometry by means of dual laser excitation

Summary A dual laser beam excitation device for flow analysis of biological particles has been developed. The aid of this arrangement is to increase the range of fluorescent agents employed so far in quantitative and qualitative cytochemistry. Combining an argon ion and a helium-cadmium laser two color fluorescence measurements were performed employing propidium iodide as a DNA stain and fluorescamine which stains total protein in fixed cells. Energy transfer processes between the antibiotic and DNA specific dye mithramycin and propidium iodide both being bound to nuclear chromatin were analyzed. Utilization of energy transfer processes is generally discussed as a mean to extract information about the structure and conformation of nuclear chromatin in situ. The application of a crypton ion laser with three lines near 400 nm and a single line at 350 nm having a light output in each range of nearly one Watt gives the opportunity of utilizing DNA fluorochromes which have an excitation maximum in the deep blue region. DNA spectra are shown employing mithramycin, the benzimidazol derivative 33258 (Hoechst) and the indol compound DAPI which has a high DNA specifity combined with a great stability under UV illumination. By separating two focussed laser beams at their intereecting points with the liquid sample stream the trajectory of each flowing cell crosses the beams sequentially, which causes a solitary dual excitation of each cell. The advantages of a solitary excitation device compared with a simultaneous one is discussed.This work has been supported by the ministry of research and technology (FRG), contract No. 01VH015-B13MT 225a     

Application of an optical immersion-gel in a flow cytometer with horizontally oriented objective

In certain flow cytometry systems, it is desirable to use immersion optics to obtain optimum fluorescence yield. This is important when propidium iodide and other DNA fluorochromes are used that have weaker fluorescence emission compared to DAPI, when a lamp is used instead of a laser and when the DNA concentrations are low. Our Partec PA II with a horizontally oriented objective and a vertically oriented flow chamber precludes using a liquid immersion medium. The problem was solved using an optical gel with appropriate characteristics. This gel is commercially available and commonly used for connecting glass fiber cables, but has never been used for microscopy before. Compared to the manufacturer's objective (40 ×, aperture 0.8), the fluorescence yield was improved approximately four-fold using the optical gel and a 40 × glycerol objective (aperture 1.25). This innovation widens the applicability of flow cytometers with horizontally oriented objectives and vertical flow chambers. We expect it to facilitate the use of propidium iodide as a DNA stain, especially when interspecific genome size comparisons are to be done and base ratio dependent bias must be avoided.