bluuebunny/crossref_metadata_2025 huggingface dataset:
Differentiation of HL‐60 promyelocytic leukemia cells: Simultaneous determination of phagocytic activity and cell cycle distribution by flow cytometry
Authors: Blair Owen C. , Carbone Rocco , Sartorelli Alan C.
Published: August 2005

Phagocytosis of fluorescent microspheres by HL‐60 promyelocytic leukemia cells following induction of differentiation with dimethyl sulfoxide (DMSO) was monitored using flow cytometry. Initiation of phagocytic capability following initiation of differentiation with 1.5% DMSO coincided with the attainment of respiratory burst activity as measured by NBT (nitro blue tetrazolium) reduction; the degree of phagocytic activity was dependent upon parameters such as microsphere size, microsphere number, and exposure time. Ingestion of fluorescent microspheres did not interfere with the measurement of DNA content using propidium iodide; thus, simultaneous determination of phagocytic activity and the cell cycle phase was possible. Accumulation of cells in the G1/G0 phase of the cell cycle following DMSO treatment was correlated with the acquisition of the capacity to phagocytize. Analysis of two‐parameter correlated data also indicated that phagocytosis is coupled with residence in the G1/G0 phase of the cell cycle, further suggesting that the ability to phagocytize fluorescent microspheres is associated with end‐stage differentiation.
Sequential monitoring of peripheral blood lymphocyte subsets in rats
Authors: Thompson Sandy C. , Bowen Kerry M. , Burton Robert C.
Published: August 2005

The methodology of enumeration of T cell subsets in humans is well established and has widespread clinical application. Sequential monitoring of lymphocyte subsets in small animals requires a consistent and reliable methodology for staining and enumerating lymphocyte populations. The optimal conditions for enumerating subclasses of rat peripheral blood lymphocytes with the Spectrum III flow cytometer are described. The sources of biological variation and technical error that were considered in optimizing the techniques and the limitations in applying them are discussed.
Multidimensional slit‐scan detection of bladder cancer preliminary clinical results
Authors: Wheeless L. L. , Berkan T. K. , Patten S. F. , Reeder J. E. , Robinson R. D. , Eldidi M. M. , Hulbert W. C. , Frank I. N.
Published: August 2005

A multidimensional slit‐scan flow system was developed for the automated recognition of abnormal cells derived from cancer of the uterine cervix and its precursors. It provides great sensitivity in both its ability to recognize cellular abnormality and to deal with the myriad potential causes of false alarms in an automated flow system. While its initial application was the automated recognition of the spectrum of neoplasia in gynecologic cytology samples, a preliminary study was carried out using specimens obtained from the urinary bladder. Cellular material was collected by bladder irrigation and stained with the fluorochrome acridine orange. One hundred fifty‐three bladder irrigation specimens, including 115 abnormal specimens containing cells derived from neoplastic lesions of the bladder epithelium, were analyzed. For the purposes of this study, abnormal specimens from the urinary bladder included specimens containing cells derived from the following lesions of the urothelium: dysplasia (atypical hyperplasia), carcinoma‐in‐situ, and transitional cell carcinoma, grades 1–3. Approximately 50,000 cells were analyzed for most specimens. Of the 38 presumed normal specimens (specimens containing only normal urothelial components), four were instrument classified abnormal. For the 69 specimens containing cells derived from transitional cell carcinoma, grade 1, 1–2, 2, 66 were correctly classified as abnormal while three were classified as normal. There were no false negatives in the 17 specimens containing cells derived from transitional cell carcinoma, grade 2–3, 3, or in the two specimens containing cells derived from carcinoma‐in‐situ. Of the eight specimens containing cells derived from dysplasia (atypical hyperplasia), seven were instrument classified abnormal. This system has demonstrated utility in the study and recognition of the cellular components of urothelial cancer and its precursors present in the urinary bladder.
Flow cytometers using optical waveguides in place of lenses for specimen illumination and light collection
Authors: Shapiro Howard M. , Hercher Michael
Published: August 2005

Optical waveguides represent alternatives to lenses for delivery of light to, and collection of light from, small regions of space such as the observation point(s) of a flow cytometer. The flow chamber and directly associated illumination and collection optics of a flow cytometer can be built using fiber optic waveguides in fixed positions or as an integrated optical assembly incorporating deposited or implanted waveguides, providing illumination and collection geometries and efficiencies comparable to conventional designs, with the advantage that the optical waveguide design, once assembled, never again requires alignment of the optics. A prototype apparatus in which fiber optics were used to illuminate and collect light from a point inside a thin‐walled, round quartz capillary was observed to measure fluorescence of polystyrene spheres with precision close to that obtained from a conventional flow cytometer using the same laser source. The design of the fiber optic system is readily adaptable to construction of multiple illumination beam instruments; other optical waveguide flow cytometer designs that dispense with the capillary share this advantage and can also provide orifices for electronic volume sensing and for jetting prior to droplet sorting.
Denaturation, renaturation, and loss of DNA during in situ hybridization procedures
Authors: Raap A. K. , Marijnen J. G. J. , Vrolijk J. , van der Ploeg M.
Published: August 2005

With the aim of optimizing in situ hybridization methods, alkaline, acid, and thermal denaturation procedures have been studied for their ability to separate the DNA strands of nuclear DNA and for the DNA losses they induce. Isolated methanol/acetic acid‐fixed mouse liver nuclei have been used as a biological object. The results, obtained with acridine orange staining and microfluorometry, show that all denaturations studied lead to almost complete strand separation. Quantitative DNA staining and cytometry indicated that with heat and alkaline denaturation about 40% of the DNA is lost. Acid denaturation led to about 20% DNA loss. For the alkaline denaturation, the DNA retention could be improved to a 20% DNA loss by adding 70% ethanol to the denaturation medium. During hybridization, another 20% DNA loss occurs.

When denatured nuclei are brought under annealing conditions, a rapid renaturation of a considerable fraction of the remaining DNA occurs. The extent of renaturation was dependent on the type of denaturation used. For the ethanolic alkaline denaturation, it was estimated to be 35%. Quantitative nonautoradiographic in situ hybridization experiments with acetylaminofluorene‐modified mouse satellite DNA showed that alkaline denaturation procedures are superior to the heat and acid denaturation.

As proven by acridine orange fluorescence measurements, hybridization conditions can be designed that permit DNA. RNA hybridization under in situ DNA. DNA denaturing conditions. These conditions should be very useful, especially for in situ hybridization with single‐stranded RNA probes.
Chemosensitivity of human small cell carcinoma of the lung detected by flow cytometric DNA analysis of drug‐induced cell cycle perturbations in vitro
Authors: Engelholm Svend A. , Spang‐Thomsen Mogens , Vindeløv Lars L. , Brünner Niles A.
Published: August 2005

A method based on detection of drug‐induced cell cycle perturbation by flow cytometric DNA analysis has previously been described in Ehrlich ascites tumors as a way to estimate chemosensitivity. The method is extended to test human small‐cell carcinoma of the lung. Three tumors with different sensitivities to melphalan in nude mice were used. Tumors were disaggregated by a combined mechanical and enzymatic method and thereafter have incubated with different doses of melphalan. After incubation the cells were plated in vitro on agar, and drug induced cell cycle changes were monitored by flow cytometric DNA analysis. Melphalan produced a dose‐related S phase accumulation in the two sensitive tumors, whereas no changes in the cell cycle distribution were found in the resistant tumor. The size of S phase accumulation correlated to the chemosensitivity in vivo. For low concentrations of melphalan, the S phase accumulation was accompanied by G2 + M accumulation. The results indicate that the method may be extented to sensitivity testing of human solid tumors, including screening for new agents.
Graphical presentation of multidimensional flow histogram using hexagonal segmentation
Authors: Kosugi Yukio , Ikebe Jun , Shitara Nobuyuki , Takakura Kintomo
Published: August 2005

Improvements in the method of presenting a multidimensional histogram for flow cytometric data are described. Two‐dimensional data were systematically quantized into hexagonal subareas, which facilitated an accurate presentation of the distribution with a smaller number of bins and sample cells.
Cell‐cooling in flow cytometry by peltier elements
Authors: Göttlinger Christopher , Meyer Klaus L. , Weichel Walter , Müller Werner , Raftery Brian , Radbruch Andreas
Published: August 2005

We have built a cooling device for cell suspensions in flow cytometry that makes use of the Peltier effect (Barnard RD, Thermo electricity in Metals and Alloys, Taylor and Francis, London; Siemens‐Z 34:383–88, 1963). The prototype described here is used for cooling collection tubes during long‐duration cell sorting and is capable of maintaining a temperature of 2–5°C in a cell suspension of up to 3 ml. In general, Peltier element‐based cooling is useful for equilibrating the temperature of small volumes of fluids. Furthermore, Peltier element‐based cooling devices are easy to build and handle.
A comparison between flow cytometric ploidy investigation and chromosome analysis of 32 human colorectal tumors
Authors: Petersen Steffen E. , Friedrich Ursula
Published: August 2005

The correlation between flow cytometric ploidy investigation and classic chromosome analysis was studied in 32 human colorectal tumors. Flow cytometry was performed by nuclei isolation and DNA staining with ethidium bromide. Chromosome analysis was done after incubation with colcemid. In 12 cases, chromosome identification was possible by grouping according to the Denver system or by Q‐banding. Generally, the measured DNA content corresponded well with the content expected from chromosome analysis, giving an average difference of 4%. In nine tumors, the measured DNA content was 4‐18% higher than expected. Some of these discrepancies could be due to difficulties in identifying the corresponding cell populations in heterogeneous tumors. However, in general the number of cell populations and their quantitative representation by the two methods were statistically well correlated.

The results indicate that flow cytometric ploidy investigation of colorectal tumors with the present technique is a reliable method, but also that a combination of both techniques may yield additional information about tumor cytogenetics.
Growth and analysis of vascular smooth muscle on microspheres
Authors: Dennis Patricia A. , Wolley Robert , Taylor Nancy S. , Moyer Carolyn F.
Published: August 2005

Protocols for culturing vascular smooth muscle cells (VSMCs) on polystyrene microspheres, assessing VSMC contractility while adhered to the microspheres, and analyzing VSMCs cultured on microspheres by flow cytometry are described. These procedures preserve optimal VSMC viability and function, while providing a method for assessing these cells within the parameters required for analysis by flow cytometry.
A quantitative study of the development of type II pneumocytes in fetal lung
Authors: Leary J. F. , Finkelstein J. N. , Notter R. H. , Shapiro D. L.
Published: August 2005

Cell populations dissociated from fetal rabbit lungs were analyzed by laser flow cytometry for the presence of type II penumocytes. These cells are distinguishable by the staining of their lamellar bodies with the fluorescent lipophilic dye, phosphine‐3R and by their intensity of low‐angle light scatter. Lung cells were obtained by enzymatic dissociation from fetal rabbits at gestational ages of 24 d, 27 d, and from 2‐d newborn rabbits. Flow cytometric analysis was sufficiently sensitive to discriminate between fetuses. Quantitative analysis of type II pneumocytes showed that newborn rabbits had a distinct cell subpopulation in a region of low‐angle light scatter and phosphine‐3R fluorescence intensity similar to that previously reported on type II cells from adult rabbits. By contrast, 24‐d gestation rabbits had a negligible type II cell subpopulation. Fetuses of 27 and 30 d gestation showed a slow but progressive increase in the numbers of cells in the type II region. Mathematical analyses of light scatter and fluorescence intensity distributions were used to define statistically significant (P < .05) boundaries that characterize the development of the type II cell subpopulation in fetal rabbit lung. The methods employed offer new possibilities for quantification of developing lung cell subpopulations of particular interest to the problem of respiratory distress syndrome in human neonates.
A method for determining ploidy distributions in liver tissue by stereological analysis of nuclear size calibrated by flow cytometric DNA analysis
Authors: Danielsen Håvard , Lindmo Tore , Reith Albrecht
Published: August 2005

A method is presented for determining ploidy distributions in mouse liver from image analysis with stereological estimations of nuclear size in tissue sections. Nuclear profile distributions obtained from profile measurements were subjected to a mathematical unfolding procedure in order to obtain the nuclear size distributions. Based on the assumption that nuclear size increases monotonically with nuclear DNA content, flow cytometric DNA analysis of suspensions of liver cell nuclei was used to calibrate the method, thus yielding the mean nuclear size of each ploidy class, i.e., diploid, tetraploid, and octaploid nuclei. After the size interval for each of the ploidy classes was determined, the method allowed determination of ploidy distributions in mouse liver by stereological image analysis alone.

The method was established from combined stereological and flow cytometric measurements on liver tissue representing two different stages of liver regeneration after two‐thirds partial hepatectomy, and it was tested against an independent set of data representing a marked increase in the portion of S‐phase cells.
Dual‐laser, differential fluorescence correction method for reducing cellular background autofluorescence
Authors: Steinkamp John A. , Stewart Carleton C.
Published: August 2005

A method has been developed for reducing the intrinsic autofluorescence background component in cells labeled with fluorescent antibodies, thus permitting low levels of antibody‐binding on highly autofluorescent cells to be quantified. The method is based on the broad autofluorescent excitation spectra compared to the well‐defined spectra of the fluorescent label. Two laser wavelengths were used, one optimally to excite the fluorescent label plus autofluorescence and the second to excite only the autofluorescence. Two fluorescence measurements were made in the same wavelength region and the signals were subtracted on a cell‐by‐cell basis using a difference amplifier to zero the autofluorescence and amplify the signal from the fluorescent label. Test results on unlabeled autofluorescent macrophages showed that the autofluorescence component was reduced by balancing the signal inputs to the difference amplifier. When labeled macrophages were analyzed, the autofluorescence was reduced and the fluorescent‐labeled antibody‐binding component was amplified. The method was also able to resolve labeled lymphocytes from unlabeled autofluorescent macrophages.
Mapping by chromosome sorting of several gene probes, including C‐myc, to the derivative chromosomes of a 3;8 translocation associated with familial renal cancer
Authors: Harris Peter , Morton Cynthia C. , Guglielmi Paul , Li Frederick , Kelly Kathleen , Latt Samuel A.
Published: August 2005

In eight members of a single family a constitutional translocation t(3;8) (p14.2;q24.1) is associated with the development of renal cancer. Chromosomes isolated from a cell line established from a subject with this translocation were analysed in flow with a fluorescence‐activated cell sorter (FACS II). Nearly six million chromosomes from the flow karyotype region containing the der(8) and 5.5 million from the region containing the der(3) were sorted, the DNA extracted, digested with EcoRI, size fractionated by electrophoresis, and transferred to nitrocellulose. Hybridization with gene probes for c‐mos, which has been localized to 8qll‐q22 and so‐matostatin, which has been mapped to 3q28, confirmed that the sorted fractions contained, respectively, the der(8) and der(3) chromosomes. The cellular oncogenes c‐raf‐1 (3p25) and c‐myc (8q24) were found to be translocated to the der(8) and der(3) chromosomes, respectively. The possible role that the relocation of c‐myc might have on the development of renal carcinoma in carriers of this 3;8 translocation was further studied by analysis of the region surrounding the c‐myc gene. By the use of cosmid cloning, no rearrangement 31 Kb 5′(or 19 Kb 3′) of the translocated gene was found, indicating that the breakpoint is not immediately adjacent to c‐myc.

In an associated study, the DNA fragment D3S2 from chromosome 3 was found to map to 3p14.2‐pter. This assignment in conjunction with published somatic cell hybrid data enabled D3S2 to be mapped more precisely to the interval 3p14.2–3p21.
Extraction of nuclei from selected regions in paraffin‐embedded tissue
Authors: Oud Peter S. , Hanselaar Ton G. J. M. , Reubsaet‐Veldhuizen José A. M. , Meijer Jos W. R. , Gemmink Anita H. , Pahlplatz Martin M. M. , Beck Hans L. M. , Peter Vooijs G.
Published: August 2005

A method for the extraction of nuclei from selected regions in paraffin‐embedded tissue is described. Fifty‐micrometer sections are cut, dewaxed, and rehydrated. For the final handling, the sections are manually transferred from one tray to another. The sections are put on a slide under a dissection microscope and the region of interest is isolated by scraping off the irrelevant region with a scalpel. An optimal number of single nuclei is obtained by incubation in a protease solution with intermediate syringing. The nuclei are washed and can be used for flow cytometry. Resuspension of the nuclei in foetal calf serum and cytocentrifugation results in preparations suited for image analysis. DNA cytometric measurements of nuclei in a carcinoma in situ and an invasive carcinoma region in breast tissue present in the same tissue block and in a severe dysplasia/carcinoma in situ (CIN III) region of the cervix are presented.
A simple method to select specific tumor areas in paraffin blocks for cytometry using incident fluorescence microscopy
Authors: van Driel‐Kulker A. M. J. , Eysackers M. J. , Dessing M. T. M. , Ploem J. S.
Published: August 2005

A simple method is described for the selection of tumor areas in paraffin blocks for cytometry. The surface of a paraffin block is stained with the fluorescent dye DAPI. By means of incident fluorescence microscopy on the cut surface of a total block, the tissue can be visualized. Location of the tumor area with the aid of conventional histopathological criteria is feasible using the adjacent section after hematoxylin‐eosin staining as a diagnostic guideline. Once the position of the tumor area is determined, a small hollow bore, which is screwed in the objective holder, is pressed in the tissue. The depth is controlled by the microscrew of the microscope. After retraction of the bore, the block is removed, and a thick section is cut. The selected area can be processed for cytometry separately from the remaining tissue. The technique can be used either to enrich the sample to be analyzed with tumor cells or to analyze histopathologically different tumor compartments. Both flow and image cytometry can make use of this selection technique.
Representing cell cycle data
Authors: Ubezio Paolo , Capolongo Laura
Published: August 2005

In this communication we suggest two simple ways to represent the information on the cell cycle obtained by flow cytometry, offering some advantages over the traditional plots. We show that the results of a single experiment, when reduced to the percentages of cells in G1, S, and G2M phases, can be completely expressed with a single point in a G1–G2 plan where iso‐S lines are also drawn. By the use of this plot, the time course of the complete kinetics of a cell population may be shown in a single drawing. The second plot, we suggest, integrates information on cell cycle, represented by the distribution mean, with cell number.
A pragmatic approach to the analysis of DNA histograms with a definable G1 peak
Authors: Watson James V. , Chambers Stephen H. , Smith Paul J.
Published: August 2005

A method for DNA histogram analysis is described that depends only on the simple assumption that the data are normally distributed and a requirement that a G1 peak is present. A probability density function was derived from the assumption that extracted the S‐phase component from the whole histogram. The model was tested with simulated data, and good agreement between predicted and known proportions in G1, S, and G1+M was found. Good agreement was also found between duplicates of experimentally derived data. Some systematic errors are present in the analysis of certain types of histograms. However, these result in small errors when compared with biological and experimental variation and are less than the average of algorithms in current use.

The program required only two queued requests, those of the start and the end channels over which the analysis is to be performed. The algorithms perform rapidly on a micro‐computer with only 28K addressable memory. Only two failures occurred in over 350 analyses and the method can be used for drug‐ and radiation‐perturbed populations as well as with unperturbed.
Effects of hyperthermia, irradiation, and cytotoxic drugs on fluorescein isothiocyanate staining intensity for flow cytofluorometry
Authors: Dyson J. E. D. , McLaughlin J. B. , Surrey C. R. , Simmons Deena M. , Daniel Jill
Published: August 2005

Measurement of fluorescein isothiocyanate (FITC) staining intensity of cultured lymphoblastoid cells following hyperthermia showed large increases without concomitant increases in nuclear protein. Similar measurements of cells following incubation with cytotoxic drugs showed fluorescent intensity increases that exceeded the increases in nuclear protein that were due to the cell cycle blocking action of the drug. The reverse, however, was true for cells following irradiation. In contrast, FITC staining intensity and nuclear protein measurements of cells proceeding through the cell cycle after removal of the cycle blocking agent showed nearly parallel changes, although there were reproducible minor differences, especially following blocking with hydroxyurea. These results suggest that FITC staining intensity is a function not only of nuclear protein content but also of stain access to the reaction sites of the protein constitutents of the chromatin. Thus, it is possible that FITC staining may be used as a probe of changes in chromatin structure following experimental manipulation of cells in vitro or treatment of tumors in vivo.
A flow cytometric study of DNA staining in situ in exponentially growing and stationary Euglena gracilis
Authors: Bonaly J. , Bre M. H. , Lefort‐Tran M. , Mestre J. C.
Published: August 2005

DNA stainability by different fluorochromes has been compared in exponentially dividing and stationary Euglena cells. With the intercalating fluorochromes, ethidium bromide, acridine orange and DAPI, a decrease of fluorescence intensity of the G1 cells is observed when cells enter stationary stage. However this decrease of fluorescence is not obtained with the nonintercalating fluorochrome Hoechst 33258. If nuclear basic proteins are extracted, however, the intensity of staining by either Hoechst 33258 or ethidiumbromide is comparable in stationary and dividing cells. Therefore, the decrease of fluorescence intensity of the G1 cells observed during the transition from exponential to stationary phase is not due to a loss of DNA but is related to the exposure of chromatin binding sites for ethidium bromide. In Euglena cells, DNA accessibility for intercalating fluorochromes depends upon chromatin structure and consequently upon cell age.
DNA flow cytometry of control Euglena and cell cycle blockade of vitamin B12‐starved cells
Authors: Lefort‐Tran M. , Bre M. H. , Pouphile M. , Manigault P.
Published: August 2005

Vitamin B12 starvation in Euglena induces a cell cycle arrest that leads to unbalanced growth. Microfluorometry and flow cytometry analyses of cellular DNA fluorescence after Hoechst 33258 staining were performed on control and vitamin B12‐deficient cells. Convergent results are obtained with both methods. Histograms that represent arrested cells are unimodal, with a mode channel value nearly twice that of the G1 control cell peak. Dispersion of fluorescence values is great, and values from 2C and over 4C are observed and discussed. It appears that vitamin B12 starvation in Euglena leads to defective DNA synthesis. Blocked cells have different DNA content, corresponding to blockade of DNA replication during the S phase. A second block prevents the onset of mitosis even for 4C cells.
Comparison of helium‐neon and dye lasers for the excitation of allophycocyanin
Authors: Loken Michael R. , Keij Jan F. , Kelley Keith A.
Published: August 2005

Allophycocyanin (APC) has a broad absorption spectrum permitting several different lasers to be used to excite this dye in a flow cytometer. A comparison was made between a dye laser and a helium‐neon (HeNe) laser for the excitation of APC as an immunofluorescent chromophore. The ratio of fluorescence of stained to unstained lymphocytes (signal to background) was used to assess differences in sensitivity. In determining the best wavelength for operating the dye laser, it was found that there was little difference in the ability to separate the positive‐labelled cells from the unstained cells using 600 nm or 633 nm light for excitation of APC. A study of the effect of laser power on the signal to background identified a nonlinear relationship. It was found that the sensitivity obtained with 47 mW of 633 nm light from a HeNe laser was near the maximum attainable. This sensitivity was comparable to that obtained using phycoerythrin as an immunofluorescence chromophore. APC had the added advantage of being applicable to the study of highly autofluorescent cells. Exciting this chromophore using red light dramatically decreased the autofluorescence observed even on alveolar macrophages.
Closed loop television tracking of beating heart cells in vitro
Authors: Schuette W. H. , Burch C. , Roach P. O. , Parrillo J. E.
Published: August 2005

The motion of beating heart cells in vitro has been used as a sensitive indicator of the presence of drugs. The combination of closed loop television video tracking and latex microspheres placed on the cells as markers results in an improved measurement of cell motion. Latex microspheres, 5.2 micrometers, are allowed to settle onto beating my ocardial heart cells in vitro, where they move in concert with the cell motion. These microspheres, when viewed by televised transmission microscopy with normal optics, present high contrast circular targets that are excellent for closed loop television video tracking techniques. With video tracking, an analog voltage is generated proportional to the horizontal center of intensity of the target being tracked. This signal represents the location of a video demodulator gate that is maintained centered over the video target by feedback control.
A single laser method for subtraction of cell autofluorescence in flow cytometry
Authors: Alberti Saverio , Parks David R. , Herzenberg Leonard A.
Published: August 2005

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, autofluorcscence 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 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.
Trout and salmon erythrocytes and human leukocytes as internal standards for ploidy control in flow cytometry
Authors: Iversen O. E. , Laerum O. D.
Published: August 2005

Control of technique and use of biological standards in flow cytometry have become increasingly important due to the wider use of the method for ploidy determination of malignant tumors in clinical research. Trout (TRBC) and salmon erythrocytes and human buffy coat leukocytes were selected for a study of factors influencing the DNA stainability. Whether standard and test cells were mixed before or after enzymatic treatment and staining was found to be critical for the ploidy comparisons. Otherwise, artifactual differences of at least 20% may be noted, leading to an overestimation of DNA aneuploidy. The time from staining to analysis had minimal effect, with some exceptions. The proportions of different cells in the sample had no influence, and nonlinearity of measurements was negligible. Diploid cells in normal endometrium and benign ovarian tumors, as well as the diploid fraction of aneuploid tumor cells, were systematically measured to have a DNA staining 5–7% above human leukocytes.
Cytophotometric determination of unscheduled DNA synthesis
Authors: Bose Kallol K. , Allison David C.
Published: August 2005

We describe a cytophotometric assay for unscheduled DNA synthesis (UDS) in asynchronously growing cells. Monolayer cultures of human HEp–2 and mouse MCa–11 cells were incubated with the carcinogen methylmethane sulfonate (MMS), as well as with hydroxyurea and (3H)thymidine. Slides were prepared, and the DNA contents and areas of nuclei were measured by absorption cytophotometry. The labeling of the nuclei, determined on the basis of their DNA content to be in G0/G1, was selectively measured after the preparation of autoradiographs. The labeling of the G0/G1 cells increased with increasing doses of MMS. We also found that the increased nuclear labeling after MMS treatment was not due to induction of replicative DNA synthesis or selective destruction of G0/G1 cells. The results of this assay compared favorably with a standard biochemical method for measuring unscheduled DNA synthesis by benzoylated naphthoylated DEAE cellulose chromatography.
An improved immunocytochemical procedure for high‐sensitivity detection of incorporated bromodeoxyuridine
Authors: Beisker Wolfgang , Dolbeare Frank , Gray Joe W.
Published: August 2005

This report describes an improved immunochemical procedure to stain cells in suspension for incorporated bromodeoxyuridine (BrdUrd) and total DNA content. The procedure consists of five steps: (1) chromatin proteins are extracted by treating with 0.1 M HCl and 0.7% Triton X‐100 to facilitate DNA denaturation and to minimize nonspecific staining; (2) cellular DNA is denatured by heating to 100°C in distilled water; (3) BrdUrd in single‐stranded DNA (ssDNA) is stained using an immunochemical procedure; (4) autofluorescence is reduced using sodium borohydride (NaBH4); and (5) DNA is stained with the fluorescent dye propidium iodide. With this procedure, the BrdUrd incorporated by CHO cells during periods as short as a few seconds can be detected using flow cytometry. In addition, the stoichiometry of the immunofluorescent staining procedure is high.
DNP‐phycobiliproteins, fluorescent antigens to study dynamic properties of antigen‐IgE‐receptor complexes on RBL‐2H3 rat mast cells
Authors: Seagrave Jean Clare , Deanin Grace G. , Martin John C. , Davis Bruce H. , Oliver Janet M.
Published: August 2005

In RBL‐2H3 rat mucosal mast cells, the crosslinking of cell‐surface IgE‐receptor complexes by multivalent antigens initiates a sequence of responses leading to degranulation. We have developed a family of dinitrophenol (DNP)‐conjugated fluorescent antigens to study dynamic membrane events associated with these responses. Lysyl groups on the phycobiliproteins, B‐phycoerythrin and C‐phycocyanin, were labelled with DNP, yielding fluorescent conjugates that cause the release of [3H]serotonin from anti‐DNO‐IgE‐primed RBL‐2H3 cells. The binding of these antigens to IgE‐receptor complexes was observed by fluorescence microscopy and quantified by flow cytometry. Incubation with 1 μg/ml DNP42‐B‐phycoerythrin stimulates maximum degranulation from IgE‐saturated cells. Under these conditions, approximately 26 × 103 molecules of DNP42‐B‐phycoerythrin are bound per cell at equilibrium. The rate and extent of antigen binding and of antigenstimulated mediator release decrease in parallel as the concentration and DNP:protein ratio of the fluorescent conjugates is reduced. Secretion stops immediately when the nonfluorescent monovalent antigen, DNP‐lysine, is added to degranulating cell suspensions. DNP‐lysine also displaces surface‐bound antigen when added during the first minutes after multivalent antigen. However, the ability of DNP‐lysine to displace surface‐bound DNP42‐B‐phycoerythrin from IgE‐receptor complexes decreases progressively with time. Treatment with dihydrocytochalasin B and several analogs that prevent antigen‐stimulated F‐actin assembly enhances secretion and delays the transition of antigen to its DNP‐lysine‐resistant form. Cytochalasin treatment also permits the long‐range movement of antigen into surface caps. Based on these data, we propose that secretion is triggered by the act of IgE‐receptor crosslinking or by a shortlived excited state of the crosslinked antigen‐IgE‐receptor complex. We propose further that antigen‐stimulated F‐actin assembly contributes to the transition of antigen‐IgE‐receptor complexes to a DNP‐lysine‐resistant form that does not trigger secretion. Two posible mechanisms for the transition to DNP‐lysine resistance are discussed.
Patterns of anthracycline retention modulation in human tumor cells
Authors: Krishan Awtar , Sridhar Kasi S. , Davila Enrique , Vogel Charles , Sternheim William
Published: August 2005

Laser excitation of cellular doxorubicin and daunomycin content (with or without incubation in the presence of efflux blockers such as phenothiazines or verapamil) was studied in cells from leukemic peripheral blood, bone marrow aspirates, and ascites and pleural fluid of solid tumor patients. Selected examples are presented to show that heterogeneity in cellular anthracycline retention as well as sensitivity to efflux modulations is seen in human tumor cells. Several tumor subpopulations differing in their cellular retention of anthracyclines or sensitivity to modulators were seen. In serial tumor samples from patients (pre‐ and post‐treatment with anthra‐cycline‐containing protocols), initial drug retention andsensitivity of efflux modulators was followed by lack of drug retention and insensitivity to modulators. The present study shows that in view of the variables encountered in drug‐retention characteristics and sensitivity to efflux modulators, one needs to screen tumor samples before recommending use of any particular transport modulator to enhance drug retention and sensitivity of drug‐resistant cells.
Quantification of epithelial volume by image processing applied to ovarian tumors
Authors: Schipper N. W. , Smeulders A. W. M. , Baak J. P. A.
Published: August 2005

This paper describes an image analysis technique for automated estimation of the percentages of epithelium and stroma in (tumor) tissue. The program is evaluated on ovarian tumors of the serous, mucinous, and endometrioid type. From standard paraffin sections, stained with pararosanilin Feulgen and naphthol yellow, a blue‐yellow image pair was recorded. The blue image was used for the determination of the total tissue area and the yellow image for the epithelial area. For the latter the image processing method is based on the fact that epithelial nuclei are generally more tightly packed than stromal nuclei. A structural approach was applied, in which the segmentation of the nuclei was based on the image contrast range in thedensity domain. The method has been tested with 78 image pairs from 19 ovarian tumors with varying degrees of malignancy. The area percentages, as assessed with image processing, were strongly correlated to control percentages, established by interactive morphometry (r = 0.98).
A modular detector for flow cytometric multicolor fluorescence measurements
Authors: Steinkamp John A. , Habbersett Robert C. , Stewart Carleton C.
Published: August 2005

A modular detector for measuring multicolor fluorescence from cells illuminated by single or multiple lasers has been developed for flow cytometers. Motion picture projector, camera, and CCTV/video lenses were evaluated for use in the detector by comparing their physical characteristics, image quality, and light collection efficiencies. A 25‐mm focal length F/0.95 CCTV lens was selected, based on our critera and test results. The detector was constructed out of square aluminum extrusion channels. A CCTV lens mounted on the outside of the first channel collected light emitted from cells and collimated it towards filters and secondary CCTV lenses located in each channel. The secondary lenses functioned as relay optics for directing and focusing light onto pinhole spatial filters for measurement by photomultipliers. The detector design allowed any number of channels to be connected together and the versatility for making simultaneous or sequential measurements. Measurements on lymphocytes labeled with four monoclonal antibodies conjugated to fluorescent dyes and measurements on viable tumor cells stained for DNA content and with three fluorescent‐labeled antibodies were used to demonstrate the detector's capabilities.
Use of tetrameric antibody complexes to stain cells for flow cytometry
Authors: Wognum Albertus W. , Thomas Terry E. , Lansdorp Peter M.
Published: August 2005

Bifunctional tetrameric complexes of monoclonal antibodies were used to stain cells for flow cytometry. These complexes consist of two different mouse monoclonal IgG1 antibodies (one with specificity for a cell surface antigen, the other with specificity for a fluorochrome) cross‐linked by two molecules of a monoclonal rat anti‐mouse IgG1. The use of this immunological approach to cross‐link fluorochromes to cell surface antigens was studied with tetrameric complexes containing Leu‐3a or Leu‐2a antibodies and monoclonal antibodies specific for the fluorochromes B‐ and R‐phycoerythrin. The ability of such cyclic immune complexes to stain T‐cell subset antigens on human peripheral blood lymphocytes was demonstrated in single and double‐staining experiments. The results demonstrate that tetrameric antibody complexes provide a simple and efficient alternative to covalently labeled antibodies for the flow cytofluorimetric analysis of cell‐surface antigens.
An improved method for the immunocytochemical detection of bromodeoxyuridine labeled nuclei using flow cytometry
Authors: Schutte B. , Reynders M. M. J. , van Assche C. L. M. V. J. , Hupperets P. S. G. J. , Bosman F. T. , Blijham G. H.
Published: August 2005

A new staining protocol is described for the immunocytochemical detection of BrdUrd labeled nuclei. Pepsin treatment of ethanol fixed cells or tissue, followed by DNA denaturation at low pH, resulted in (1) increased sensitivity of BrdUrd staining comparable to the thermal denaturation protocol, and (2) decreased background binding. This technique is applicable to cell suspensions, including cultured cells and bone marrow cells. Furthermore, pepsin digestion of ethanol fixed tissue fragments resulted in a high recovery of nuclei in which incorporated BrdUrd could be detected. This possibility, together with the high sensitivity, make this method especially suitable for cell kinetic studies of human solid tumors in vivo.
Flow cytometric analysis of murine splenic B lymphocyte cytosolic free calcium response to anti‐IgM and anti‐IgD
Authors: Chused Thomas M. , Wilson H. Alexander , Greenblatt David , Ishida Yasuo , Edison Linette J. , Tsien Roger Y. , Finkelman Fred D.
Published: August 2005

The accurate measurement of ionized intracellular calcium [Ca++]i in single cells by flow cytometry with the use of a new fluorescent calcium chelator, indo‐1, is described. We have developed a dependable in situ calibration technique that indicates a resting [Ca++]i in lymphocytes of 100 nM. The enhanced fluorescence of this probe permits its use at sufficiently low cytoplasmic concentrations that buffering of [Ca++]i transients does not occur.

The [Ca++]i response of small resting B lymphocytes to crosslinking of surface antigen receptors by anti‐immunoglobulin is heterogeneous. With maximal stimulus, the peak [Ca++]i response occurs in 10 to 20 seconds with most cells reaching levels >/1 μM. Mean [Ca++]i falls to between 300 and 800 nM by 100 seconds where it remains for more than 10 min. Anti‐δ is a more potent stimulus of increased [Ca++]i than anti‐μ in terms of both [Ca++]i level and fraction of B cells responding. Whether this is due to the greater density of surface IgD than IgM, a difference in signal transduction efficiency, or both, is not yet known. Surface immunoglobulin receptors are present in great excess. Less than 3% of surface immunoglobulin is crosslinked at the peak of the [Ca++]i response.
Anomalous behaviour of forward and perpendicular light scattering of a cyanobacterium owing to intracellular gas vacuoles
Authors: Dubelaar George B. J. , Visser Jan W. M. , Donze Marcel
Published: August 2005

Extinction, absorption, and forward and perpendicular light scatter of the blue‐green alga Microcystis aeruginosa with different amounts of intracellular gas vacuoles were determined. The amount of gas vacuoles in the cells was controlled by application of pressure. The presence of the gas vacuoles caused a tenfold increase in perpendicular light scatter, and a fivefold decrease in forward light scatter as measured by flow cytometry. Chlorophyll fluorescence showed a 16% decrease. The presence of gas vacuoles did not affect the size of the algae. The absorption spectrum of Microcystis aeruginosa was slightly raised but practically not distorted by the gas vacuoles. The attenuation spectrum, a measure for light extinction by the algal cells, was significantly distorted. The increase of perpendicular light scatter intensity of the cells is a direct consequence of the relatively high scatter of each vacuole, whereas the forward light scatter decrease is attributed to a lower phase‐shift factor s̀ of the whole cells, caused by the intact gas vacuoles.
Discrimination of viable and non‐viable cells using propidium iodide in two color immunofluorescence
Authors: Sasaki Dennis T. , Dumas Stephanie E. , Engleman Edgar G.
Published: August 2005

The relative ease with which a flow cytometer can perform simultaneous two color immunofluorescence to examine subpopulations of lymphoid cells has been well documented. Thus, flow cytometers equipped with only a single argon laser can be used to delineate various cell types by exciting both fluorescein‐ and phycoerythrin‐conjugated antibodies to cell surface antigens. One problem that remains, however, is the artifactual staining of dead cells and clumps, which cannot be distinguished from viable cells on the basis of cell surface staining characteristics. We describe a method for simultaneous two color analysis or sorting of viable leukocytes which requires only a single laser. The method utilizes propidium iodide, which stains dead cells and thereby excludes such cells from the analysis. Using this method, as many as four viable cell types have been simultaneously analyzed in a single sample.
Viable sorting of intact multicellular spheroids by flow cytometry
Authors: Freyer James P. , Wilder Mark E. , Jett James H.
Published: August 2005

A flow cytometric method has been developed for sorting viable, intact multicellular spheroids in order to obtain uniformly‐sized populations with diameters in the range of 50–100 μm. A FACS II instrument was modified for this purpose by installing a 200‐μm‐diameter exit orifice and by making adjustments in the sheath flow, oscillator frequency, and number of droplets sorted. Polystyrene microspheres (44 and 88 μm diameter) and 41–96‐μm‐diameter spheroids could be sorted and recovered with 70–100% efficiency, an improvement over previous reports. Unstained, viable spheroids were simultaneously analyzed for small‐angle forward light scatter, 90° light scatter, and autofluorescence using a 488‐nm laser operating at 100 mW. Analysis of the data demonstrated a considerable variation in both the 90° light scatter and the autofluorescence signals for a given forward angle light scattering signal. By setting narrow sort windows on the forward angle light scattering signal and either the 90° light scatter or autofluorescence signals, uniformly spherical spheroid populations could be recovered. These sorted populations had coefficients of variation of the mean diameter in the range of 5–9%. This represents a variation of less than one cell diameter, and is a major improvement over any other technique. There was no significant difference in the subsequent growth rates of sorted spheroids compared to the unsorted spheroids. This technique will apply when uniform populations of small spheroids are required, such as in investigations of the contact effect or in the initiation of growth curve studies.
A comparison of low‐power helium‐cadmium and argon ultraviolet lasers in commercial flow cytometers
Authors: Bigler Robert D.
Published: August 2005

The feasibility of installing a low power ultraviolet (UV) laser in a commercial flow cytometer was evaluated by testing an Ortho Cytofluorograf 50HH and a Coulter Epics V. Both instruments were equipped with two argon ion lasers, one emitting at 488 nm and the other in the UV region and were tested by measuring the DNA content of cells stained with Hoechst 33342 or DAPI. The coefficient of variation (CV) of the G0/G1 peak of the DNA histograms produced by each instrument did not deteriorate markedly when results obtained at 100–125 mW were compared to those obtained at 10 mW. These tests indicated that a helium‐cadmium laser (He‐Cd) which can produce 10 mW at 325 nm should work well as a UV laser in these instruments. An Ortho Cytofluorograf IIs was purchased with a 10 mW He‐Cd laser installed in the forward position. Studies of DNA content have confirmed that this low power UV laser can produce CVs of 2.2% with DAPI stained fixed cells and 3.6% with Hoechst 33342 stained viable lymphocytes. Thus, the He‐Cd laser should provide a reasonable alternative as a UV source for flow cytometers.