bluuebunny/crossref_metadata_2025 huggingface dataset:
Centromeric index versus DNA content flow karyotypes of human chromosomes measured by means of slit‐scan flow cytometry
Authors: Lucas J. N. , Gray J. W.
Published: August 2005

We have applied slit‐scan flow cytometry (SSFCM) to classify human chromosomes according to their centromeric index (CI) and relative DNA content. The resulting bivariate—CI vs. DNA content—distributions shows 14 peaks for normal human chromosomes. Distinct peaks are produced by chromosomes 1, 2, 3, 4+5, 6+7+X, 8, 13+14+15, 16, 17+18, 19+20, and 21+22+Y. In addition, chromosomes 9 through 12 are resolved into three peaks. The identity of the chromosomes comprising each peak was determined by comparing CI vs. DNA content distributions measured for normal human chromosomes by means of SSFCM with CI and DNA content values measured for human chromosomes with image analysis. The accuracy of CI measurement by SSFCM was verified by measuring CIs for human chromosomes isolated from human/rodent hybrid cell lines containing only a few known human chromosomes. These studies showed CIs measured for human chromosomes 1–19 and 21 to be inclose agreement with the CIs calculated by means of image analysis. We further confirmed the chromosome assignments for each peak by showing that the relative volumes of the peaks in the CI vs. DNA content distributions for chromosomes from normal cells are similar to the relative frequencies of chromosomes expected for these peaks based on the peak assignments.
Quantitative analysis of a nuclear antigen in interphase and mitotic cells
Authors: Clevenger Charles V. , Epstein Alan L. , Bauer Kenneth D.
Published: August 2005

The quantification of an interchromatin‐as‐sociated antigen, designated p105, during cellular passage through mitosis is described. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated a qualitative increase in p105 within the mitotic cytoplasm. Multiparameter flow cytometric analysis was performed on fixed cells sequentially stained with anti‐p105 immunofluorescence and/or propidium iodide. This analysis demonstrated approximately a tenfold increase in intracellular p105 content as a function of progression from the G2 to the M phase. This increase was corroborated by the quantitative immunoblot analysis of colchicine‐treated cell cultures and of cells sorted on the basis of anti‐p105 immunofluorescence. The data reveal that the increased levels of anti‐p105 immunofluorescence in conjuction with flow cytometry may be used effectively to quantitate mitotic index and isolate mitotic cells. The function and modulation of p105 throughout the cell cycle is discussed.
Comparison of synchronized Chinese hamster ovary cells obtained by mitotic shake‐off, hydroxyurea, aphidicolin, or methotrexate
Authors: Fox Michael H. , Read Robert A. , Bedford Joel S.
Published: August 2005

Synchronized cell populations are necessary to study many aspects of cell biology. We have developed a method to obtain highly synchronized Chinese hamster ovary cell populations in S phase or G2 phase by utilizingmitotic selection followed by incubation with either hydroxyurea, aphidicolin, or methotrexate for 12 h. Flow cytometry analysis shows that the coefficient of variation in the spread of the cell population in S phase is as low as 6%. Drug toxicity studies compare the effects of the various drugs on G1 and S phase cells. The use of aphidicolin or hydroxyurea results in the most highly synchronized cell populations, but methotrexate yields inadequate synchronization. These results demonstrate that both aphidicolin and hydroxyurea are useful drugs for obtaining highly synchronized cell populations after an initial synchrony in mitosis. Aphidicolin is perhaps the best choice because of less toxicity to S phase cells when used in low concentrations.
Monoclonal antibody isolation of type II pneumocytes
Authors: Funkhouser Jane D. , Cheshire L. Bryan , Read Robert J. , Hester Raymond B. , Peterson Raymond D. A.
Published: August 2005

A monoclonal antibody that identifies a membrane molecule unique in rat lung for type II alveolar epithelial cells was used to isolate these cells from enzymatically dispersed lung cells by fluorescence‐activated cell sorting. Although multistep physical separation techniques have permitted the isolation of large quantities of these cells and flow cytometry has been used by others to isolate lamellar body‐containing cells, the application of this antibody‐directed sorting has distinct advantages. Because the marker molecule is expressed on immature type II cells prior to the development of lamellar bodies, the antibody will also permit their isolation and study.
Improved flow cytometry of cellular DNA and RNA by on‐line reagent addition
Authors: Pennings Arie , Speth Paul , Wessels Hans , Haanen Clemens
Published: August 2005

Acridine orange (AO) staining enables flow cytometric measurement of cellular DNA and RNA. With the conventional procedure, red and green fluorescence intensities alter considerably in time, affecting the reliability and reproducibility of the results. A standardized simple technique of AO staining is described that keeps the time between addition of detergent and AO and the subsequent measurement constant. Measurements by means of this on‐line method appear to be much more reproducible in comparison to those obtained using the conventional procedure.
Multiple wavelength illumination in flow cytometry using a single arc lamp and a dispersing element
Authors: de Grooth B. G. , van Dam M. , Swart N. C. , Willemsen A. , Greve J.
Published: August 2005

The principle of a multiple wavelength illumination method for flow cytometers, based upon a combination of a helium‐neon laser and an arc lamp as illumination sources is described. By using a prism, the light from the arc lamp is dispersed and the different colors are imaged at different places on the sample stream. The small angle light scattering from the helium‐neon laser light is measured as a relevant parameter and serves as a trigger signal for subsequent measurements of fluorescence or scattering of light from the arc lamp. Two experimental systems are described utilizing this principle: a system where the emission is detected orthogonally with respect to the direction of the illumination beams, and an epi‐illumination system.

With the orthogonal set‐up multiple wavelength right angle scattering measurements are possible. This is illustrated by showing that the orthogonal scattering from erythrocytes is strongly dependent on the illumination wavelength. It is further shown that the apparatus is suitable for the measurement of intracellular pH using the pH dependence of the excitation spectrum of fluorescein.

The epi‐illumination system allows excitation of two (or more) fluorescent dyes with different excitation spectra. In this case the emission spectra of the fluorescent dyes may overlap substantially. This is shown by simultaneous measurement of DNA and protein of Chinese hamster lung cells using mitramycin and tetramethyl rhodamin isothiocyanate (TRITC).
Evaluation of DNA and nuclear protein features for their use in describing normal and malignant endometrium
Authors: Oud Peter S. , Katzko Michael W. , Pahlplatz Martin M. M. , Vooijs G. Peter
Published: August 2005

Different feature sets (geometric, densitometric, and textural) derived from DNA and nuclear protein staining were evaluated for their use in describing atrophic, secretory, and proliferative endometrium, and well‐differentiated stage I and moderately differentiated stage I adenocarcinomas of the endometrium. It was found that the pattern of significant differences among these groups varied between feature sets, while remaining consistent within a set of features. The DNA density and run‐length features were not very effective in describing group mean differences, whereas the co‐occurrence features revealed significant differences among most groups. The protein run‐length features were the only ones that consistently showed a difference between proliferative endometrium and well‐differentiated adenocarcinomas. Analyses repeated on only cells in the G0/G1 DNA region improved the differentiation between moderately differentiated adenocarcinomas and the other groups. It was concluded that the use of DNA and nuclear protein texture features are effective in describing group differences that cannot be described by DNA content only.
Visualization of DNA loops in nucleoids from HeLa cells: Assays for DNA damage and repair
Authors: Roti J. L. Roti , Wright W. D.
Published: August 2005

An assay for visualization of DNA loops undergoing supercoiling changes has been developed. The assay utilizes the fluorescent dye, propidium iodide (PI), which intercalates into the DNA and under the proper conditions causes the supercoiling status of the DNA to change. Thus, the DNA can be seen as a fluorescent halo that changes diameter with PI concentration. At low PI concentrations (0–7.5 μg/ml) the supercoils are relaxed with increasing PI, while at higher PI concentrations (7.50–50 μg/ml) supercoils in the opposite winding sense are rewound with increasing PI. When HeLa cells were irradiated with 1–20 Gy of 137Cs γ‐rays, the ability to rewind the DNA supercoils was inhibited in a dose‐dependent manner, presumably because of the presence of radiation‐induced DNA strand breakage, which removed the topological constraints on the DNA loops. These lesions were repaired rapidly during post‐irradiation incubation. The ability of the DNA loops to be rewound was restored within 8 min after 10 Gy of γ‐irradiation, such that no difference from control cells could be detected. The halftime for repair of the radiation‐induced lesions that inhibit DNA rewinding was similar to that for repair of DNA single strand breaks. The assay has certain advantages over current methods for assaying DNA damage in that it involves measurement of single cells and it does not require the DNA to be labeled with radioactive precursors.
Quantification of the nonspecific intercellular transfer of fluorescent molecules between labeled and unlabeled rat thymocytes
Authors: Capo Christian , Mege Jean Louis , Benoliel Anne Marie , Mishal Zohair , Bongrand Pierre
Published: August 2005

Fluorescein isothiocyanate has been used to label normal or tumor cells in order to study their migration in vivo. This requires that any transfer of fluorescence to neighbouring cells be carefully ruled out. The aim of the present report is to demonstrate the possibility of a transfer of fluorescein or fluorescein‐bound molecules between untreated and labeled cells. When normal rat thymocytes were co‐incubated with labeled cells (about 5 × 106 fluorescein molecules/cell) under continuous agitation or exposed to supernatants of these labeled cells, they bound an average of 104 fluorescein molecules. When the incubation was done on cell pellets after centrifugation, this transfer was increased tenfold. Hence, intercellular molecule exchange may occur in the absence of any specific interaction.
Flow cytometric analysis of DNA in cells obtained from deparaffinized formalin‐fixed lymphoid tissues
Authors: McIntire Timothy L. , Goldey Stacia H. , Benson Neal A. , Braylan Raul C.
Published: August 2005

Flow cytometric analysis of DNA content was performed on nuclear suspensions prepared from fresh and from paraffin‐embeded, formalin‐fixed lymphoid tissues. We confirmed previous reports that it is possible to obtain nuclear suspensions from deparaffinized, formalin‐fixed tissues, suitable for DNA analysis by flow cytometry. We observed a tendency for a larger coefficient of variation (CV) of the DNA measurements in the fixed tissues than in the unfixed material causing abnormalities in 2 of 19 lymphomas to become undetectable. Furthermore, samples from different paraffin blocks of a single tumor with an extra G1 (hyperdiploid) peak showed marked differences in the CV of the hyperdiploid peak while the CV of the diploid peak was similar in all samples. In both benign and malignant lymphoid tissues, the S‐phase fraction was higher in paraffin‐embedded tissues than in unfixed cells. This difference could be attributed to 4′, 6′‐diamidino‐2 phenylindole dihydrochloride (DAPI), a DNA‐binding dye commonly used in this technique. Nevertheless, intermediate and high grade lymphomas from paraffin‐embedded tissues generally showed a greater S‐fraction than low grade lymphomas, a similar observation as with unfixed tissues. Therefore, DNA content analysis of nuclei extracted from paraffin sections may be inadequate to resolve slight aneuploidy, but the measurement of S‐fraction size may remain diagnostically or prognostically valuable. Large retrospective studies will be necessary to determine the clinical impact of this technique in the analysis of lymphomas.
Surface glycoproteins of differentiating neuroblastoma cells analyzed by lectin binding and flow cytometry
Authors: Notter Mary F. D. , Leary James F.
Published: August 2005

Cell surface glycoprotiens of mitotic neurobalstoma cells and cells differentiated by prostaglandin cyclic adenosine monophosphate treatment were quantified by flow cytometric analysis and specific fluorescent lectins. No differences in fluorescent lectin binding were seen between suspensions of mitotically active and differentiated N2AB‐1 cells following exposure to either fluorescein (FL)‐labeled soy bean agglutinin (FL‐SBA) specific for N acetyl galactosamine or FL‐concanavalin A (FL‐CON A) which binds to mannose residues. These lectins, however, were shown to bind specifically to these cells as revealed by competitive blocking studies with hapten sugars. When FL Ulex europaeus (FL‐UEA) specific for fucose was reacted with control or differentiated cells, no binding was seen even with an increased dose of lectin before or after enzyme treatment. However, differentiated N2AB‐1 cells, reacted with FL‐wheat germ agglutinin (FL‐WGA) specific for N acetyl glucosamine, bound more FL‐WGA than that seen for control cultures. Furthermore, specific sites for FL‐WGA were shown to be saturable and were lost upon pretreatment of cells with neuraminidase. Neuraminidase pretreatment revealed masked sites for FL‐CON A and FL‐SBA since binding was increased at least twofold for these lectins on mitotic and differentiated cells. These data indicate that single cell measurements of surface glycoproteins can be made on living neural cells and that differentiation induces an increase in cell surface N‐acetyl glucosamine residues.
A rapid and simple estimation of cell cycle parameters by continuous labeling with bromodeoxyuridine
Authors: Sasaki Kohsuke , Murakami Tomoyuki , Takahashi Manabu
Published: August 2005

The DNA synthesis time (Ts) and other related cell cycle parameters were roughly estimated in HeLa cells labeled with bromodeoxyuridine (BrdUrd) for various durations by using the flow cytometrical technique. The labeling indices increased in proportion to time after addition of BrdUrd. The Ts can be calculated from the slope of the regression line obtained by plotting the serial labeling indices against the labeling time and was equivalent to the value determined by fraction labeled cells in mid S‐phase (FLSm) method. These parameters would be determined by only two samples labeled for different times. This simple method using BrdUrd provides rough but rapid estimation of Ts and other cell cycle parameters without complicated mathematical procedures, in addition to cell cycle partition of cell populations.
Technique and staining optimization leucoconcentration
Authors: Pierrez J. , Guerci A. , Guerci O.
Published: August 2005

In cytometric clinical application, it is important to obtain cell suspensions rapidly with as little cytological alteration as possible. A procedure has been achieved to prepare cell suspensions for flow cytometric analysis. The leucoconcentration technique, first described by Herbeuval for cytologic analysis, has been modified to be applied in cytometry. This technique involves Saponin lysis of red cells of peripheral blood or bone marrow samples that have been previously fixed with picric acid alcohol solution. Cells in suspension are not shifted and tinctorial affinity is not modified.

Then cells have been stained with Mithramycin. Each parameter defined by Crissman has been analyzed to define the best staining conditions.

The availability of Leucoconcentration with Mithramycin‐DNA‐staining permits determination of cell cycle with a fine resolution.
Thiazole orange: A new dye for Plasmodium species analysis
Authors: Makler M. T. , Lee L. G. , Recktenwald D.
Published: August 2005

A rapid sensitive method for the determination of Plasmodium falciparum in in vitro culture is presented. The technique employs a fluorescent flow cytometer equipped with a 15‐mwatt argon laser that emits light at 488 nm and a membrane‐permeable fluorochrome thiazole orange (TO) that stains RNA. Parasitized red cells are stained by suspending them in 1 ml of phosphate‐buffered saline (PBS) containing 10×5 M of TO and incubating this mixture for 15 min in the dark at room temperature. The stained cells may be analyzed fresh or after fixation with 1% paraformaldehyde/PBS or 0.25% glutaraldehyde/PBS. Alternatively the cells may be fixed first and then stained. There is excellent correspondence between the number of fluorescent‐labeled parasitized red cells and Giemsa‐stained cells.
Characterization of a carbocyanine derivative as a fluorescent penetration probe
Authors: Olive P. L. , Durand R. E.
Published: August 2005

The fluorescent carbocyanine dye 3,3‐diheptyloxycarbocyanine [DiOC7(3)], originally described as a membrane potential probe, penetrates poorly into multicell spheroids. Since the dye is retained in the cells following spheroid disaggregation, cells can be selected from different depths within the spheroid using fluorescence‐activated cell sorting. Characterization of the binding kinetics, stability, and toxicity of this probe were undertaken, and intercompared with Hoechst 33342. The optimum drug dose for achieving good separation of internal and external cells of spheroids is about tenfold lower than for Hoechst 33342, and like Hoechst, DiOC7(3) is toxic at concentrations at least tenfold higher than those required to produce a good gradient for cell separation. When cells are removed from the stain, cellular fluorescence decreases to half the initial intensity within 2 hours; however, unlike Hoechst, the carbocyanine dye does not transfer between cells.
Feulgen DNA stainability of bone tumors after demineralization
Authors: Bauer Henrik C. F. , Kreicbergs Andris
Published: August 2005

Microspectrophotometric DNA analysis of archival bone tumor tissue is often impeded by previous acid demineralization, which destroys Feulgen DNA stainability. To find an alternative to acid for prospective DNA studies of bone tumors in tissue sections, Feulgen stainability of fresh osteosarcoma specimens after demineralization in neutral EDTA was investigated. The reliability of DNA analysis of weakly Feulgen‐stained sections from archival tissue was also studied.

Demineralization of four fresh specimens in EDTA slightly reduced Feulgen DNA stainability compared to nondemineralized preparations but did not affect the determination of ploidy level. Hydrolysis tests of one diploid and one hyperploid osteosarcoma showed that the staining relationship between control and tumor cells was not altered by EDTA pretreatment. For DNA studies of bone tumors requiring demineralization, EDTA offers a means of retaining nuclear Feulgen stainability.

In 22 archival osteosarcoma specimens of varying Feulgen stainability, three different upper limits of light transmission (75, 85, and 95%) were applied to test the significance of background disturbances in relation to nuclear stain intensity. The relationship between the median total extinction of the control and tumor cell populations was not significantly affected by altering the upper transmission limit except in four poorly stained lesions. The control cells of these four specimens exhibited a median total extinction less than one‐third of the maximum encountered. The results suggest that weakly stained archival specimens can be tested for selecting those appropriate for ploidy determination.
Flow cytometric analysis of isolated rat liver nuclei during growth
Authors: Papa Stefano , Capitani Silvano , Matteucci Alessandro , Vitale Marco , Santi Patrizia , Martelli Alberto M. , Maraldi Nadir M. , Manzoli Francesco A.
Published: August 2005

The development of hepatocyte polyploidy in rats aged up to 4 months was analyzed by flow cytometry using both scatter and fluorescent parameters to distinguish DNA diploid and DNA tetraploid populations and to discriminate between parenchymal and non‐parenchymal compartments. The precise origin of each class of nuclei was assessed in whole liver homogenate using purified hepatocytes, obtained by liver perfusion followed by separation on Percoll gradient, and identifying the peaks corresponding to parenchymal nuclei. The results indicate that preparative procedures involving homogenization of the rat liver tissue caused loss of the DNA octaploid population. Data on the relative proportion of the different DNA ploidy elements during rat liver development, which are in good agreement with those observed by cell analysis by means of microspectrophotometry, indicate the usefulness of flow cytometry as a choice method for the analysis of ploidy distribution.
DNA synthesis rate and unlabeled cells with S‐phase DNA content in P388 leukemic cells in vivo studied by flow cytometry and cell sorting
Authors: Clausen O. P. F.
Published: August 2005

DNA synthesis kinetics of P388 leukemic cells growing in ascites form in BDF1 hybrid mice were investigated during the periods of exponential growth and growth restriction. Incorporation of tritiated thymidine, and in some instances tritiated uridine, was studied by autoradiography in cells sorted from S‐phase fractions during DNA flow cytometry.

During exponential growth continuous labeling with tritiated thymidine indicated a growth fraction of unity, whereas the growth fraction was about 30% during growth restriction. At this growth phase the majority of cells with S phase DNA content remained unlabeled after pulse labeling with tritiated thymidine or uridine, indicating that both the “salvage” and the “de novo” DNA synthesis pathways were blocked in most S‐phase cells.

After pulse labeling with tritiated thymidine the DNA synthesis rate pattern was investigated by sorting of consecutive fractions of cells throughout the S phase followed by quantitative autoradiography. With exception of a reduced rate in the middle of S phase, the DNA synthesis rate increased as the cells progressed through S phase during exponential growth. In contrast, the DNA synthesis rate pattern had a relative peak in the middle of S phase during growth restriction, which is otherwise characterized by a low mean DNA synthesis rate.
Bivariate flow karyotyping of acute myelocytic leukemia in the BNML rat model
Authors: Arkesteijn G. J. A. , Martens A. C. M. , Jonker R. R. , Hagemeijer A. , Hagenbeek A.
Published: August 2005

Univariate as well as bivariate flow karyotyping has been performed on chromosome suspensions obtained from the Brown Norway myelocytic leukemia (BNML), a rat model for human acute myelocytic leukemia (AML). Flow karyograms were obtained from both the in vivo transplantable parent line and from an in vitro established cell line. Density gradient centrifugation performed on cells arrested in mitosis resulted in an enrichment of mitotic cells. Furthermore, with this procedure leukemic and nonleukemic cells could be separated. Univariate analysis with propididum iodide (PI) as a DNA stain revealed the position of the several tumor‐specific marker chromosomes in the in vitro cell line. Estimations of the peak position of the various chromosomes was done by comparing the univariate flow karyogram with a computer‐simulated karyogram from the BNML that was derived from the mean length of the individual chromosomes in conventionally prepared metaphase slides. By comparing the bivariate flow karyogram of the in vivo BNML cells with the flow karyogram of normal BN cells, it was clearly demonstrated which peaks are involved in the altered chromosomal pattern of the BNML. No differences were found between the flow karyograms of the in vitro‐and the ex vivo‐derived chromosome suspensions in this rat leukemia model.
Quantitative description of nuclear morphology in assessing resistance of sarcoma 180 to adriamycin
Authors: Komitowski D. , Sonka J. , Schmitt B. , Muto S.
Published: August 2005

Resistance to adriamycin generally is explained through changes of cell/drug interactions that possibly reflect structural alterations of intracellular targets. One of the main targets of adriamycin is believed to be nuclear chromatin. In order to recognize chromatin alterations, we studied cell nuclei morphology and chromatin structure by means of digital image analysis. The studies were performed in both adriamycin‐sensitive and ‐resistant Sarcoma 180 cell lines which were cultured under growth‐stimulated and nonstimulated conditions. Using specially developed methods, we extracted parameters characterizing geometrical, optical, and structural properties of the cell nuclei from light microscopical images. The latter parameters concerned microscopical appearances of condensed chromatin and were described by features of high‐optical‐density regions. The results demonstrated that the quantitative criteria applied enabled the discrimination of sensitive and resistant cells. The most important parameters are the nuclear size, number, distribution, and optical density of condensed chromatin regions. In addition, the criteria permit recognition of changes related to differences in the growth conditions of the cells. The data of the image analysis suggest that adriamycin resistance in Sarcoma 180 cells is associated with characteristic patterns of cell nuclear morphology which can be described with a sufficient number of appropriate parameters. The advantages of image analysis are evident when these results are compared with the flow cytometric findings. The conclusion is that structural features of nuclear chromatin provide information essential for the assessment of drug resistance.
Effect of drug efflux blockers on vital staining of cellular DNA with hoechst 33342
Authors: Krishan Awtar
Published: August 2005

The present study shows that staining of certain live cells, e.g., adriamycin‐resistant P388 cells, by Hoechst 33342 is difficult because of the presence of a rapid efflux pump, which reduces intracellular dye concentration. Coincubation of these refractory cells in the presence of efflux blockers such as phenothiazines (trifluoperazine) or Ca++ channel blockers (verapamil) enhances dye retention and thus leads to generation of normal DNA distribution histograms. Laser flow cytometric data is confirmed by fluorometric assays, which show that P388/R cells retain one‐third the amount of Hoechst 33342, and coincubation with efflux blockers increases Hoechst retention to values similar to those of drug‐sensitive P388 cells. DNA histograms of mouse splenocytes incubated with Hoechst 33342 alone have a bimodal distribution possibly because of the presence of subpopulations that do not retain the fluorochrome owing to rapid efflux. Coincubation with an efflux blocker results in the generation of unimodal DNA histograms from these cells.

These preliminary studies suggest that reduced retention of Hoechst 33342 in certain cell types (because of rapid efflux) can be blocked by efflux blockers, thus leading to generation of typical DNA distribution histograms.
Time, a quality‐control parameter in flow cytometry
Authors: Watson James V.
Published: August 2005

Time has been used as a quality‐control parameter in our flow cytometer. This parameter was automatically incorporated into the list‐mode data base by hooking the computer clock directly into the acquisition logic. Quality control can then be checked by “replaying” fluorescence or light scatter data vs. time and any instrumental drift can be observed and degraded data can be excised. The technique is illustrated with a number of DNA data sets from a tissue culture cell line in which artificial perturbations were introduced during data acquisition to simulate potential causes of instrumental drift.
Condensation of DNA in situ in metaphase chromosomes induced by intercalating ligands and its relationship to chromosome banding
Authors: Darzynkiewicz Zbigniew , Kapuscinski Jan
Published: August 2005

Interactions of certain intercalating cationic ligands with nucleic acids result in the formation of products that undergo condensation and agglomeration; this transition in solution can be monitored by light‐scatter measurements. In the present study, using such intercalators as the antitumor drug mitoxantrone or fluorochromes acridine orange and quinacrine, we induced condensation of DNA in situ in Chinese hamster chromosomes. The in situ products scattered light and could be detected by darkfield‐ or phase‐contrast microscopy. In the darkfield the complexes had a characteristic granular appearance and often generated a banding pattern on the chromosomes. In contrast, condensation of DNA in situ by the nonintercalating polyvalent cations (Co3+, spermine4+), while enhancing the chromosome's image contrast, did not produce the granular products or the banding.

The condensation of free DNA, single or double stranded, natural or synthetic, the later of various base composition and configuration, was also measured in solution. The condensation in solution and in situ was observed at similar concentrations of the respective ligands. The intercalating dye ethidium bromide, which did not condense DNA in solutions of moderate and high ionic strength, also did not generate the granular products or banding on chromosomes. The data also show that both base composition and configuration are important factors in determining the sensitivity of DNA to condensation by particular intercalating ligands.

The studies suggest that the phenomenon of DNA condensation by intercalating dyes, which shows a high degree of specificity with respect to primary and secondary structures of DNA, may be associated with mechanisms of chromosome banding induced by the intercalating thiazine dyes in Giemsa staining or by quinacrine. Observation of chromosome banding based on light‐scatter detection in darkfield microscopy allows the study of interactions between DNA and the ligands that neither fluoresce nor generate colored products. This principle of chromosome “counter‐staining” can be explored by flow cytometry.
Light scattering and cell volumes in osmotically stressed and frozen‐thawed cells
Authors: McGann Locksley E. , Walterson Michele L. , Hogg Lori M.
Published: August 2005

Recent reports, indicating that under some conditions the intensity of light scattering from cells is a nonlinear function of cell volume, have led to the widespread generalization that intensity of low‐angle light scattering indicates cell size. This study was performed to measure the relationships between light scattering and cell volumes in anisotonic solutions and after a freeze‐thaw stress.

Cell volumes in isolated human lymphocytes, human granulocytes, and hamster fibroblasts were deliberately altered by exposure to anisotonic solutions. Boyle‐vant Hoff plots of cell volume as a function of inverse osmotic pressure showed that the cells behaved as osmometers. Similar plots of right‐angle and low‐angle light scattering showed that the intensity of light scattering varied inversely with cell volume.

In other experiments where cells were frozen without cryoprotectant at various sub zero temperatures to −25°C and then thawed rapidly, cell viability decreased progressively with decreasing temperature, as did the intensity of both low‐angle and right‐angle light scattering, although cell volumes remained relatively constant.

The intensity of both low‐ and high‐angle light scattering varied inversely with cell volumes in hypertonic and hypotonic solutions, but cell damage induced by freezing and thawing resulted in significant reductions in the intensity of low‐angle light scattering with little change in cell volume.

These observations show that light scattering and cell volumes can vary independently, and they underline the need for a better understanding of the phenomenon of light scattering from living cells.
Nonselective enrichment for yeast adenine mutants by flow cytometry
Authors: Bruschi Carlo V. , Chuba Paul J.
Published: August 2005

The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade 1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence‐activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.
Detergent treatment of dictyostelium discoideum cells allows examination of internal cell type‐specific antigens by flow cytometry
Authors: Bernstein R. L. , Browne L. H. , Yu S. C. , Williams K. L.
Published: August 2005

Monoclonal antibodies are used extensively in flow cytometry to identify subpopulations of cells differing in surface antigens. Conventional studies on living cells do not allow analysis of internal antigens, because antibody molecules do not pass through an intact plasma membrane. It is important for developmental studies on Dictyostelium discoideum that not only surface but also internal antigens be analysed. Here techniques are reported that make possible such studies by permeabilising cells with mild detergent treatments using digitonin. Flow cytometer profiles of unfixed cells show that antigens recognised by two monoclonal antibodies, MUD102 and MUD3, are found inside subpopulations of cells in the D. discoideum slug. Double‐labelling experiments were carried out to demonstrate that the antigens recognised by these antibodies are present inside prespore but not prestalk cells. The detergent treatment leads to loss of forward‐angle light scatter, but 90° light scatter of cells is not greatly affected. While fixed cells sometimes gave satisfactory results, internal labelling did not reliably demonstrate the two subpopulations observed with unfixed cells.
Fringe‐scan flow cytometry
Authors: Mullikin J. , Norgren R. , Lucas J. , Gray J.
Published: August 2005

We describe the development of a scanning flow cytometer capable of measuring the distribution of fluorescent dye along objects with a spatial resolution of 0.7 μm. The heart of this instrument, called a fringe‐scan flow cytometer, is an interference field (i.e., a series of intense planes of illumination) produced by the intersection of two laser beams. Fluorescence profiles (i.e., records showing the intensity of fluorescence measured at 20 ns intervals) are recorded during the passage of objects through the fringe field. The shape of the fringe field is determined by recording light scatter profiles as 0.25 μm diameter microspheres traverse the field. The distribution of the fluorescent dye along each object passing through the fringe field is estimated from the recorded fluorescence profile using Fourier deconvolution. We show that the distribution of fluorescent dye along microsphere doublets and along propidium iodide stained human chromosomes can be determined accurately using fringe‐scan flow cytometry. The accuracy of fringe‐scan shape analysis was determined by comparing fluorescence profiles estimated from fringe‐scan profiles for microspheres and chromosomes with fluorescence profiles for the same objects measured using slit‐scan flow cytometry.
Flow cytometric detection of herpes simplex virus type 2 infected and transformed cells by immunoenzymatic and by indirect immunofluorescence staining
Authors: Goolsby Charles , Gay Helen , Docherty John J. , Todd Paul
Published: August 2005

The glucose oxidase antiglucose oxidase (GAG) immunoenzymatic staining procedure has been used to detect herpes simplex virus (HSV) antigens microscopically. In this study, the GAG procedure was adapted to cells in suspension, and its potential usefulness in flow cytometry was examined. HSV‐2 infected monkey kidney and HSV‐2 transformed mouse cells were stained using antisera to HSV‐2 or to an HSV‐2 specific protein with a molecular weight of 38 Kd, respectively, with the GAG procedure. Flow cytometric analysis of the GAG stained cells was then performed by the measurement of scattered light intensity in the angular intervals l°−2°, 2.5°−19°, and 3°−6°. The greatest scattered light intensity decrement caused by staining occurred in the 3°−6° angular interval, as predicted by previous work. In infected cells, which stain intensely by immunofluorescence, the difference between positively and negatively stained cells was adequate for detecting infected cells using the GAG method; however, this was not the case for the lightly staining transformed cells. The indirect immunofluorescence method of analysis of the same populations was superior to the scattered light method of analysis of the GAG stained infected and transformed cells.
High‐speed photodamage cell selection using bromodeoxyuridine/Hoechst 33342 photosensitized cell killing
Authors: Herweijer Hans , And Willem Stokdijk , Visser Jan W. M.
Published: August 2005

One of the major drawbacks of droplet sorting in a flow cytometer is the relatively low sorting speed. Thus, we have developed an alternative, faster sorting technique: photodamage cell sorting. In a photodamage cell sorter all unwanted cells, as detected with the first, measuring laser, are killed with the second, damaging laser. Thus, the cells need to be photosensitive to the second laser. In addition, a mechanism is needed to switch this laser on and off based on the sorting criteria. In our photodamage cell sorter, the ZAPPER, we use an acousto‐optic crystal to switch the laser beam. Cells are made photosensitive by vital staining with photosensitizers. With cells grown in the presence of 5‐bromo‐2′‐deoxyuridine (BrdUrd) and stained with Hoechst 33342 (H42) at least a 5‐decade cell reduction is accomplished after irradiation with 400 mW UV light. With this system, sorting rates have been achieved of 30,000 cells per second. Due to the selection based on photodynamic killing, this sorting technique is restricted to the selection of viable cells.

Photodamage cell sorting seems well suited for isolating viable cells occurring in low percentages or for the sorting of large numbers of cells. Another application can be the sorting of large or fragile cells.
Flow cytometric measurement of pollutant stresses on algal cells
Authors: Berglund Deborah L. , Eversman Sharon
Published: August 2005

The lichen Usnea fulvoreagens (Räs.) Räs. was treated with four pH levels (5.5, 4.5, 3.5, and 2.5) of simulated acid rain (sulfuric acid, nitric acid, and a 1:1 combination of both) and automobile exhaust. The samples were dissociated and analyzed by a Becton‐Dickin‐son FACS 440 flow cytometer. Analyses included measurement of chlorophyll autofluorescence and fluorescence due to uptake of fluorescein diacetate (FDA) and calcofluor white M2R (CFW). Cell parameters measured were esterase activity (FDA), membrane permeability (FDA, CFW), and intracellular pH (FDA). Mean fluorescence intensity from FDA staining and numbers of events were incorporated with autofluorescence information to produce a “stress index” of relative cell stress. Results indicated that highly stressed samples (lower pH treatments and greater exposure to exhaust) exhibited a low “stress index” of FDA fluorescence.
Cyclosporin a does not inhibit the PHA‐stimulated increase in intracellular Ca2+ concentration but inhibits the increase in e‐rosette receptor (CD2) expression and appearance of interleukin‐2 receptors (CD25)
Authors: Redelman Doug
Published: August 2005

The immunosuppressive drug cyclosporin A (CsA) inhibits mixed lymphocyte responses, blocks the generation of cytotoxic T lymphocytes, and inhibits the T lymphocyte proliferative response stimulated by polyclonal activators such as phytohemagglutinin (PHA). Nevertheless, there have been contradictory reports attempting to explain the mechanism(s) for this immunosuppressive activity. In the current studies, human peripheral blood mononuclear cells (PBM) were stimulated with PHA in the presence or absence of CsA. Flow cytometric examination of PBM loaded with the Ca2+‐sensitive dye Indo‐1 showed that concentrations of CsA sufficient to inhibit 90‐100% of tritiated thymidine incorporation had no effect on the PHA‐stimulated increase in the intracellular Ca2+ concentration ([Ca2+]i). Likewise, inhibitory amounts of CsA had virtually no effect on the increase in cell volume that occurs during T lymphocyte activation. These results were not altered by pretreating the PBM with CsA for 30 min at 37°C prior to adding the PHA. On the other hand, inhibitory concentrations of CsA prevented the expression of receptors for T cell growth factor (interleukin‐2, IL‐2), as measured by monoclonal antibodies to CD25 after 16‐24 hr incubation. In like manner, CsA also prevented the increase in the expression of the E‐rosette receptor (CD2) on these same cells. If cultures containing PHA and inhibitory amounts of CsA were incubated for 40‐72 h, there was partial recovery both of proliferative activity and of the expression of CD25 and CD2. Thus, CsA does not appear to affect the initial activation signal(s), but does interfere with one or more subsequent events necessary to initiate the appearance of “activation antigens.”
Cell cycle distribution in‐vivo of mononuclear blood cells of renal allograft recipients
Authors: Walle Alexander J. , Wong George Y.
Published: August 2005

In 47 healthy blood donors (controls) and 29 renal allograft recipients (patients) the relative contents of RNA and DNA of peripheral blood mononuclear (PBM) cell populations were estimated from the intensities of green and red fluorescences emitted by complexes that form with DNA and RNA, respectively, after staining the cells with the metachromatic dye, acridine orange. Based on the correlated DNA and RNA estimates for large numbers of cells, the percentages and the relative RNA contents of cells in particular compartments of the cell cycle were determined. PBM cell populations of controls contained < 0.5% proliferating (SG2M) cells with highly variable relative RNA contents. Among controls, neither percentages nor relative RNA contents of SG2M‐cells were correlated with percentages or relative RNA contents of G1‐cells with an RNA content 2 (2SD) or 3 (3SD) standard deviations above the mean of the entire GO/1‐cell population. Unlike controls, PBM cell populations of patients contained significantly higher percentages of SG2M‐cells which were significantly correlated with the relative coefficients of variation (rCV) of the F530 histograms of GO/1‐cells; the rCV represents the ratio of a patient's CV to a control's CV. Moreover, significant statistical correlations existed between percentages and relative RNA contents of 2SD‐, 3SD‐, SG2M‐and GO/1‐cells, suggesting a well‐orchestrated progression of cells through the cell cycle. Different pairs of correlated parameters characterized clinically stable, acutely rejecting, and infected patients. Finally, the data of this pilot project suggest that two features of GO/1‐cells, namely the rCV and the mean relative RNA content may carry the information necessary to predict the cell cycle distribution of PBM cells of stable, rejecting, or infected renal allograft recipients.
Altered nucleic acid contents of mononuclear cells in blood of renal allograft recipients
Authors: Walle A. J. , Wong G. Y. , Suthanthiran M. , Rubin A. L. , Stenzel K. H. , Darzynkiewicz Z.
Published: August 2005

The DNA and RNA contents of blood mononuclear cell populations of 29 cadaver renal allograft recipients and 49 blood donors (controls) were estimated by acridine orange flow cytometry (AO FCM) to assess their cell cycle status. All patients received azathioprine and prednisone for immunosuppression. The patients represented three clinical categories: clinically stable patients, those with acute rejections (clinically overt or impending), and those with infections. Three cell cycle compartments were analyzed for percentage (%) and RNA content (R) of cells: GO/1, consisting of all cells with diploid DNA content; 2 S.D., consisting of cells with diploid DNA content and RNA content 2 standard deviations above the mean RNA content of controls; and SG2M, consisting of cells with a DNA content higher than that of GO/1 cells. The relative coefficient of variation (rCV) of the DNA distribution of GO/1 cells was also determined. In such cell cycle evaluations, the means of rCV and SG2M% of stable recipients were significantly different from those of blood donors. Multivariate analysis of the variables of the three categories of patients resulted in the tentative formulation of two simple logistic equations: one that differentiates stable patients from those with impending or overt rejections based on 2SD% and another one that distinguishes infected patients from those with impending or overt rejections based on SG2M% and RG0/1.
Image analysis applied to proliferating cells in malignant lymphoma
Authors: Souchier C. , Ffrench M. , Scoazec J. Y. , Bryon P. A. , Berger F.
Published: August 2005

This study describes a method for obtaining the distribution of a given cell type in histological sections, as revealed by a specific immunological marker. Two hundred fifty‐six contiguous microscopic fields were analysed, and a cellular map was automatically constructed. The resulting images were stored on magnetic media for subsequent quantification and statistical tests. The method was applied to the analysis of 20 small cleaved‐cell lymphomas in which the monoclonal antibody Ki‐67 was used to mark proliferating cells. Clusters of proliferative cells were observed in both diffuse and nodular lymphomas. Cellularity and the percentage of marked cells were not significantly different in the two types of tumors.
Fixation and long‐term storage of human lymphocytes for surface marker analysis by flow cytometry
Authors: Lal Renu B. , Edison Linette J. , Chused Thomas M.
Published: August 2005

A method to preserve stained human lymphocytes for subsequent cell surface analysis by flow cytometry (FCM) is described. Cells stained with fluorescein isothiocyanate (FITC) and phycoerythrin (PE)‐conjugated monoclonal antibodies and then fixed in 1% paraformaldehyde, followed by extensive washing and resuspension in 1% BSA medium, could be stored at 4°C for at least 2 weeks prior to FCM analysis without significant alteration in the light scatter or fluorescence properties of the cells. Furthermore, the method was also suitable for analying lymphocytes that express T‐cell activation markers in certain disease conditions. In addition, we have identified monoclonal antibody combinations that discriminate different lymphocyte subsets that are satisfactory for multiparameter analysis after 2 weeks of storage. This method should prove useful for enumerating lymphocyte subsets in field study sites remote from flow cytometry laboratories.