bluuebunny/crossref_metadata_2025 huggingface dataset:
Flow cytometric analysis of liposome‐nuclei interaction: Transfer and intranuclear release of carboxyfluorescein
Authors: Papa Stefano , Caramelli Elisabetta , Allegri Giorgio , Billi Anna M. , Manzoli Francesco A. , Capitani Silvano
Published: August 2005

Flow cytometric analysis of the transfer of liposome‐encapsulated carboxyfluorescein to isolated rat liver nuclei indicated that the fluorophore is actively taken up in this form, while negligible amounts are transferred when the dye is free in the reaction medium. The kinetic analysis of the uptake indicated a time‐ and dose‐dependent reduction of the slope in the absence of transport saturable sites on the nuclear surface and of quenching phenomena. The comparison between entire and membrane‐deprived nuclei demonstrated that the initial rate of uptake was higher in the absence of the complete nuclear envelope. The intranuclear binding sites were considered on the basis of the fluorescence distribution and of quantitative estimates of the amount of linked dye. The possibility of employing flow cytometry to monitor the interactions between liposomes and isolated nuclei by means of a fluorescent probe is discussed.
Flow cytometric estimation of transmembrane potential of macrophages—A comparison with microelectrode measurements
Authors: Jenssen Hans‐Ludwig , Redmann K. , Mix E.
Published: August 2005

Potential‐dependent accumulation of the lipophilic cationic dye 3,3′ dihexyloxacarbocyanine (DiOC6(3)) in macrophages has been investigated. Resulting fluorescence of cells was measured by flow cytometry. Alterations of membrane potential of macrophages were induced by ionophore treatment (valinomycin and gramicidin) in a dose‐dependent (10−5 M‐107 M) and time‐dependent (0 min‐45 min) manner. Resulting changes in relative fluorescence intensity were compared with changes of transmembrane potential measured by intracellular recordings obtained by applying glass microelectrodes. The comparative studies offer the possibility to calibrate the flow cytometric estimate of membrane potential of suspended cells. Equilibration of dye partition between cells and surrounding medium is strictly potential‐dependent at dye concentrations between 5 × 10−8 M and 10−7 M and within an incubation interval from 10 min up to 30 min after addition of dye. Conclusions are drawn concerning the field of application of the optical method. Dynamics of electrical processes following ionophore treatment are discussed in terms of molecular mechanisms of altered ionic transport.
Flow cytometric measurement of cytoplasmic pH: A critical evaluation of available fluorochromes
Authors: Musgrove Elizabeth , Rugg Catherine , Hedley David
Published: August 2005

Three pH‐sensitive fluorochromes–4‐methyl‐umbelliferone(4MU),2,3‐dicyano‐hydroquinone (DCH), and 2′,7′‐bis(carboxyethyl)‐5,6‐carboxy fluorescein (BCECF)–were evaluated for their resolution, range, and stability of cellular fluorescence. Flow cytometric techniques for determining cytoplasmic pH (pHi) have been fully described for 4MU and DCH; BCECF has previously been used for fluorimetric estimation of pHi, and was adapted to flow cytometry. For each fluorochrome, the ratio of fluorescence intensity at two wavelengths gives a measure of pHi, which may be calibrated by obtaining the fluorescence ratios for cells suspended in buffers of varying pH in the presence of a proton ionophore.

Reliable calibration proved difficult using 4MU, partly because of poor retention within cells. Both DCH and BCECF could be calibrated using a fluorescence ratio and had resolutions of 0.2 and 0.4 pH units, respectively. The fluorescence of DCH is so strongly pH dependent that there were practical difficulties in its use over a wide pH range; however, pHi measurements are possible between pH 6.0 and pH 7.5 using either DCH or BCECF. Substantial dye leakage was found for 4MU and, to a lesser extent, DCH, while BCECF was retained by cells for up to 2 hours. Despite its lower resolution BCECF had a usable range of more than 1.5 pH units and this coupled with its stable fluorescence and excitation at 488 nm rather than UV suggests a wide application.
Analysis of intracellular antigens by flow cytometry
Authors: Jacobberger James W. , Fogleman David , Lehman John M.
Published: August 2005

Quantitative immunofluorescence is routinely used in flow cytometric assay of cell surface antigens. Intracellular antigens have not been as tractable. Recent publications (Proc Natl Acad Sci 80:5573–5577, 1983; Cytometry 6:208–214, 1985) and the results presented here demonstrate that highly specific staining and subsequent quantitative analysis are not only possible but rather easily achieved. High purity antibodies and optimized fixing and staining technique are required. Under conditions presented in this paper, 97% of fluorescein specific signal is specific to the T antigen of SV40 when monoclonal antibody to this antigen is used with a transformed cell line. Three levels of quantitative analysis are discussed: 1) estimation of the fraction of positive cells in a mixed +/− population, 2) estimation of the average content of antigen in a population of cells, and 3) measurement of the distribution of antigen content within a population of cells. Results are presented that demonstrate that relatively low specific signal (measured as percentage of total signal) can be tolerated to achieve the first level and that the current methods available that produce a high specific signal are sufficient to achieve the second level. The third level will require further research aimed at lowering the variation introduced by the method of measurement.
Leukemia‐related morphological features in blast cells
Authors: Aus H. M. , Harms H. , Haucke M. , Beritova J. , ter Meulen V. , Gunzer U. , Baumann I. , Abmayr W.
Published: August 2005

This paper investigates the use of image‐processing methods to detect leukemia‐related morphological differences in mononuclear blast cells. Routinely prepared Pappenheim‐stained blood smears were scanned in a high‐resolution color TV‐microscope system. Eleven blast‐cell classes (OMSBC, T‐ALL, OMS, ALL, LBL, IBL, AUL, AML, AMOL, AMMOL, and CML) were analyzed with the nonparametric statistical software program „Classification and Regression Trees”︁ (CART). This paper documents the initial statistical evaluation of 62 leukemia‐related morphological features that directly measure and analyze the cell‐related quantifiable differences occurring in the various blast cells. The 62 cell image features include both common cytophotometric features, and new texture and color features developed for this project. This study found that each leukemia specimen contains a dominant class of blasts that correlates with the specific leukemia, plus a distribution of blasts from related diseases. The present data suggest the existence of a distribution fingerprint pattern for each leukemia.
Can peripheral T‐Cell lymphomas be morphologically subclassified? A morphometric approach to 21 cases
Authors: Sigaux François , Flandrin Georges , Valensi Françoise , Gaulard Philippe , Castaigne Sylvie , Daniel Marie Therese , Harousseau Jean Luc
Published: August 2005

Morphometric analysis of nuclear sizes and shapes was carried out on semithin sections of lymph node for 21 patients suffering from non‐Hodgkin's peripheral T‐cell malignant lymphoma (ML) (excluding mycosis and Sézary syndrome). Twenty cases of B‐cell ML and three cases of Sézary syndrome with massive lymph node infiltration were also studied as references. Wright and Isaacson's recent proposals were applied to classify the peripheral T‐cell MLs into monomorphic medium‐cell ML (eight cases), pleomorphic ML (nine cases), and monomorphic large‐cell ML (four cases). These three classes were readily distinguishable by morphometric analysis of nuclear sizes. Nuclear areas and their coefficients of variation were higher in pleomorphic MLs than in monomorphic medium‐cell MLs (p < 0.01). Large‐cell monomorphic MLs were set apart by the histograms of their nuclear sizes. The mitoses were evaluated on histological sections and found to be more numerous in pleomorphic ML than in monomorphic medium‐cell ML (p < 0.05). Nuclear irregularity in the 21 cases of peripheral T‐cell ML was lower than in Sézary cells. Morphometry clearly demonstrates the morphological distinctiveness of the subclasses of peripheral T‐cell ML. Their biological significance has yet to be determined.
Binding, ingestion, and growth of Chlamydia trachomatis (L2 serovar) analyzed by flow cytometry
Authors: Levitt Daniel , Zable Brian , Bard Jonathan
Published: August 2005

We have developed a method for quantitatively assessing binding, ingestion, and growth of Chlamydia trachomatis (L2 serovar) in several mammalian cell lines using fluorescence staining and flow cytometry. Cells were incubated with chlamydia at 4°C to monitor binding; ingestion was determined by raising the temperature to 37°C for 1–4 h and removing extracellular bacteria with pronase. Growth of bacteria was measured by assessing brightly stained intracellular inclusions. Fixation with methanol prior to fluorescent staining provided the most intense specific staining with minimal background, as well as preserving cell morphology. Our data reveal relatively slow ingestion of L2 by McCoy fibroblasts (maximum ingestion by 4 h) and a sizeable population of McCoy cells (30–40% of total cells) that ingest L2 but do not permit its growth under certain infectious conditions. It was possible to correlate specific histogram patterns on the flow cytometer with fluorescent microscope observations. This system provides a means of analyzing quantitative interactions between chlamydia and individual host cells.
Flow cytometric estimation of cell cycle parameters using a monoclonal antibody to bromodeoxyuridine
Authors: Sasaki Kohsuke , Murakami Tomoyuki , Ogino Tetsuro , Takahashi Manabu , Kawasaki Shoji
Published: August 2005

An estimation of cell kinetic parameters was made by simultaneous flow cytometric measurements of DNA and bromodeoxyuridine (BrdUrd) contents of cells. The procedure described in this paper involves the incorporation of BrdUrd by S phase cells, labeling the BrdUrd with an indirect immunofluoresent technique using a monoclonal anti‐BrdUrd antibody, and staining DNA with propidium iodide (PI). The amount of incorporated BrdUrd in HeLa cells was proportional to that of synthesized DNA through S phase. For all cell lines examined, the pattern of BrdUrd incorporation was essentially the same and the rate of DNA synthesis during S phase was not constant. The bivariate BrdUrd/DNA distributions showed a horse‐shoe pattern, maximum in the mid S phase and minimum in the early and late S phases. Furthermore, the durations of cell cycle (Tc) and S phase (Ts) were estimated from a FLSm (fraction of labeled cells in mid S phase) curve that was generated by plotting the percentage of BrdUrd pulse‐labeled cells in a narrow window defined in the mid S phase of the DNA histogram. The values of these parameters in NIH 3T3, HeLa S3, and HL‐60 cells were in good accordance with the reported data. This FCM method using the monoclonal anti‐BrdUrd antibody allows rapid determination of both cell cycle compartments and also Ts and Tc without the use of radioactive DNA precursors.
Construction, analysis, and application to 46,XY gonadal dysgenesis of a recombinant phage DNA library from flow‐sorted human Y chromosomes
Authors: Müller Ulrich , Lalande Marc , Disteche Christine M. , Latt Samuel A.
Published: August 2005

The analysis of a recombinant human Y‐enriched Hind III total digest phage library prepared from the DNA of flow sorted human Y chromosomes is described. Out of 43 phage inserts from the library thus far mapped, 25 revealed hybridization with Y chromosomal DNA. These inserts may be divided into five groups according to their degree of Y specific hybridization: inserts that hybridize with 1) one single copy or slightly repeated Y‐specific DNA sequence, 2) Y‐specific repeated sequences of various restriction fragment lengths, 3) Y‐chromosomal DNA sequence(s) shared by a sequence on the X and/or on autosomes, 4) Y‐specific DNA sequences in addition to multiple X and/or autosomal sequences, or 5) Y‐specific repeated DNA in addition to multiple X and/or autosomal sequences. Application of probes from this library for diagnostic purposes is shown in two 46, XY patients with gonadal dysgenesis and small deletions of the Y short arm.
A method to average immunofluorescent histograms
Authors: Marti G. E. , Schuette W. , Magruder L. , Gralnick H. R.
Published: August 2005

Single parameter gated flow cytometric fluorescent histograms were obtained on normal donor blood mononuclear cells using several commonly available lymphocyte surface markers. A computer method was developed to average single parameter flow cytometric immunofluorescent histograms. The averaged histograms provide a means of pattern recognition for normal lymphocytes and will aid in the clinical evaluation of lymphocytosis. Averaged histograms may also serve as standards for more advanced analysis.
Detection of acid‐β‐galactosidase activity in viable human fibroblasts by flow cytometry
Authors: Jongkind J. F. , Verkerk A. , Sernetz M.
Published: August 2005

The fluorogenic substrate fluorescein‐di‐β‐D‐galactopyranoside was used to detect acid β‐galactosidase in intact cultured human fibroblasts. The accumulation of intracellular fluorescein, as measured by flow cytophotometry was linear with the incubation time in three control strains. The two fibroblast strains from patients with acid β‐galactosidase deficiency did not show an accumulation of intracellular fluorescence. Within one control cell population there was a positive correlation between the amount of accumulated intracellular fluorescein fluorescence and the specific acid β‐galactosidase activity as measured biochemically on sorted cells from different zones of the fluorescence distribution. No correlation was found between the specific acid β‐galactosidase activity and the fluorescein fluorescence of three different control cell strains.
Characterization of chromatin distribution in cell nuclei
Authors: Young Ian T. , Verbeek P. W. , Mayall Brian H.
Published: August 2005

In this paper we develop four measures to describe the distribution of nuclear chromatin. These measures attempt to describe in an objective and meaningful way the heterogeneity, granularity, condensation, and margination of chromatin in cell nuclei. Starting with a high‐resolution digitized image of a cell where the nuclear pixels have been identified, the four measures may be rapidly estimated. The range of each is derived and the interpretation of the measures in the context of chromatin compaction and distribution is developed. Implementation issues such as sampling density, thresholding and subsequent pre‐processing, and algorithmic complexity are discussed.
Flow cytometry reveals a high degree of genomic size variation and mixoploidy in various strains of the acellular slime mold Physarum polycephalum
Authors: Kubbies M. , Wick R. , Hildebrandt A. , Sauer H. W.
Published: August 2005

High‐resolution flow cytometry, using avian erythrocytes as an internal standard, was employed to study constitutive genome size variation of G2‐phase nuclei of Physarum polycephalum strains during the macroplasmodial stage of their life cycle. Our results document a previously unknown extent of genome size variation and mixoploidy in this organism. The unimodal diploid strain Tu 291 displayed the largest genome of the strains tested; in contrast, the Colonia strain displayed only half of the Tu 291 G2‐phase fluorescence, confirming its haploid nature. An additional strain, derived from a recent cross between Lu897 and Lu898 amoebae, must have arisen by selfing (propagation of only one of the parental genomes to the macroplasmodial stage), since its nuclei display close to the haploid G2‐phase DNA content. The observation of a small fraction of corresponding diploid nuclei within the haploid population of this strain, while maintained as microplasmodia, supports the notion that meiosis in haploid strains may require the presence of diploid nuclei. Two of the descendants of the prototype haploid Colonia strain, which were kept for extended periods of time in submerse culture, proved to be near diploid and mixoploid. Polyploidization and subsequent loss of DNA thus seems to contribute to the extremes of genome size variation in Physarum. In addition to unimodal fluorescence distributions, a number of diploid strains displayed bi‐ and even trimodal distributions within harvests of a single G2‐phase macroplasmodium. Analysis of these mixoploid strains by means of gaussian curve‐fitting suggests that the smaller genome size differences in Physarum may arise in step‐wise diminution of DNA in approximate units of 3–5% of the original Tu 291 genome.
A comment on “A method to measure the duration of DNA synthesis and the potential doubling time from a single sample”
Authors: Allen White R. , Meistrich Marvin L.
Published: August 2005

Recently a method was introduced by Begg et al. (Cytometry 6:620–626, 1985) for the calculation of the DNA synthesis time, TS using flow cytometry to monitor the progression of cells that have been pulse‐labeled by bromodeoxyuridine (BrdUrd). Ideally, the method uses only a single two‐parameter histogram of the progressed cells to obtain TS. The essence of the method is to be able to obtain an initial point from general principles, draw a line through it and the measured point, and extrapolate to a level of progression that would be attained when the time is equal to TS. In this comment, explicit values are derived for the initial point and the progression of cells based on kinetic arguments, and a new method is presented for estimating TS. This new method is compared to earlier methods, and the effects of a finite labeling time and variation in the transit times are considered. Finally, an equation is developed for estimating TS following multiple measurements.
Thiazole orange: A new dye for reticulocyte analysis
Authors: Lee Linda G. , Chen Chia‐Huei , Chiu Laura A.
Published: August 2005

The purpose of this study was to find a 488‐nm excitable fluorescent dye for reticulocyte analysis by the use of fluorescence activated cell cytometry. The chemical structure of thioflavin T, a dye used for reticulocyte analysis with 457‐nm excitation, was used as a model. Several dyes were synthesized and evaluated by quantum yield determination, fluorescence microscopy, and flow cytometry. The best results were obtained with a dye we have named “thiazole orange”; analysis of several blood samples with thiazole orange gave a correlation coefficient of 0.97 as compared to a manual determination of reticulocyte percentage.
Segmentation of stained blood cell images measured at high scanning density with high magnification and high numerical aperture optics
Authors: Harms H. , Aus H. M. , Haucke M. , Gunzer U.
Published: August 2005

In hematological morphology, it is necessary to resolve and analyze the smallest possible cellular details appearing in the light microscope. A prerequisite for computer‐aided analysis of subtle morphological features is measuring the cells at a high scanning density with high magnification and high numerical aperture optics. Contrary to visual observations, the information content in a measured picture can be increased by setting the condensor's numerical aperture (NA) greater than the objective's NA. The complexity and heterogeneity of such cell images necessitate a new segmentation method that conserves the morphological information required in the subsequent image analysis, feature extraction, and cell classification. In our segmentation strategy, characteristic color difference thresholds for each nucleus and cytoplasm are combined with geometric operations, probability functions, and a cell model. All thresholds are repeatedly recalculated during the successive improvements of the image masks. None of the thresholds are fixed. This strategy segments blood cell images containing touching cells and large variations in staining, texture, size, and shape. Biological inconsistencies in the calculated cell masks are eliminated by comparing each mask with the cell model criteria integrated into the entire segmentation process. All 20,000 leukocyte images from 120 smears in our leukemia project were segmented with this method.
Random signal variation as a cause of systematic microdensitometric error
Authors: Goldstein D. J.
Published: August 2005

A high noise/signal ratio in microdensitometry, owing, for example, to the use of a very small measuring spot or light at an extreme of the spectrum, decreases the precision (increases the variability) of results. This can be compensated for by making more measurements, but under some circumstances random signal variations can also cause systematic errors that are not so easily corrected. The present article explains how such errors can arise when measuring either very pale or very dense objects and suggests methods to detect and avoid them. The principles described are applicable to many types of microdensitometer, not only the Vickers flyingspot instruments used in this investigation.
Centrifugal separation of cells in sputum specimens from patients with undifferentiated carcinoma
Authors: Albright Craig D. , Frost John K. , Pressman Norman J.
Published: August 2005

Ethanol‐fixed cells in sputum from patients with undifferentiated carcinoma of the lung were separated in aqueous Ficoll using a discontinuous density gradient centrifugation technique. The selective enrichment of small cell undifferentiated (e.g., oat cell) or large cell undifferentiated carcinoma cells was achieved while removing most of the leukocytes (80–90%) and macrophages (65–75%) from specimen fractions containing the greatest relative frequencies of cancer cells. The maximum purity of small cell carcinoma cells (0.04%) occurs in moderate density (ρ = 1.121g/ml) gradient fractions and results in a 2.4‐fold enrichment relative to unprocessed specimens. In contrast, the maximum purity of large cell carcinoma cells (0.22%) is obtained in very high density (ρ = 1.172 g/ml) gradient fractions and results in a 1.2‐fold enrichment in comparison with unprocessed specimens. Microscopic examination of Papanicolaoustained specimen fractions reveals that these enrichments were achieved while retaining diagnostically significant cytomorphologic and tinctorial features necessary for cancer screening and diagnosis. Peak purity ranges of undifferentiated cancer cells significantly overlap comparable ranges for material from bronchogenic adenocarcinoma and squamous cell carcinoma.
The fate of the primary diploid population during spontaneous transformation of growth factor‐supplemented murine cell cultures
Authors: Kubbies M. , Friedl R. , Bettecken T. , Hoehn H.
Published: August 2005

Primary cultures derived from lung and renal tissue of the newborn harvest mouse (Micromys minutus) were serially passaged in media supplemented with epidermal growth factor, hydrocortisone, transferrin, insulin, and triiodothyronine. Although these growth factor supplements eliminated the growth crisis commonly encountered during the initial stages of murine primary cultures, the original diploid cell fraction clearly underwent such a “crisis”; the truly diploid cells invariably disappeared as these cultures reached 20 to 40 population doublings. They were replaced, either gradually or precipitously, by various heteroploid cell fractions. In three of four independent cultures, these “established” cells were hypotetraploid and appeared to be derived from a small number of progenitors already present during the very early (precrisis) culture stages. In contrast to rather frequent DNA changes displayed by clones and subclones derived from the various heteroploid cell lineages, the predominant components of the established mass cultures displayed a highly constant DNA fluorescence pattern. Our results suggest that primary murine cell cultures develop heteroploid cell lineages even if the initial growth crisis is mitigated by growth factor supplements. These heteroploid cells appear to respond more efficiently to stimulation by various growth factors than the primary diploid cell population.
Cell‐by‐cell autofluorescence correction for low signal‐to‐noise systems: Application to epidermal growth factor endocytosis by 3T3 fibroblasts
Authors: Roederer Mario , Murphy Robert F.
Published: August 2005

Autofluorescence of cells can be a major portion of the fluorescence signal in many systems, especially when fluorescent conjugates are used to study receptor‐ligand systems for which there are less than 70,000 receptors per cell. We have devised a method for the cell‐by‐cell correction of autofluorescence for flow cytometric data by using an additional parameter to measure and correct for autofluorescence in the fluorescence channel. The principle has been extended to allow simultaneous correction for autofluorescence and dual‐fluorescence spillover compensation in samples labeled with two different fluorochromes; all corrections were done in software, making them applicable to any flow cytometer. The autofluorescence correction method was used to analyze the acidification of epidermal growth factor (EGF) by Swiss 3T3 cells. EGF is acidified to pH 6.2 starting two min after labeling, with a half‐time for acidification of 45 s.
Orientation measurements of microsphere doublets and metaphase chromosomes in flow
Authors: Lucas Joe Nathan , Pinkel Daniel
Published: August 2005

Obtaining information about the shape of particles from slit‐scan profiles is facilitated if the particles are oriented. Elongated particles orient in the nozzle of flow cytometers, but orientation may be disrupted before the particles get to the point of measurement. We have used our slit‐scan flow cytometer to investigate the orientation of microsphere doublets in a liquid jet in air, in flow across a glass surface, and in a 200‐μm‐square capillary tube as a function of distance from the flow chamber nozzle. Particles were classified as being oriented if there was a centrally located dip in the slit‐scan profile. Essentially all the doublets in the jet were oriented, and no disorientation was noted over the distances measured (up to 10 mm from the nozzle). Particle orientation was maintained for 80 μm in flow across a glass surface. In the capillary‐type flow chamber, essentially all of the particles were oriented at the tube entrance and for several millimeters into the tube. There then occurred a region where particle tumbling started and progressively fewer doublets met the orientation criteria. The distance to where tumbling began could be estimated by calculating the length required to establish the parabolic flow profile in the tube. Finally, the fraction of oriented particles reached a constant value that did not change with increased distance into the tube. When sample was injected off axis (i.e., half‐way between the chamber center and the chamber walls), particle tumbling began closer to the tube entrance.

Orientation studies of Chinese hamster M3–1 metaphase chromosomes showed that over 95% of the larger chromosomes (1–5) and over 65% of the smaller chromosomes (9–11) were oriented near the entrance of the capillary tube.
Swine chromosomes: Flow sorting and spot blot hybridization
Authors: Grunwald D. , Geffrotin C. , Chardon P. , Frelat G. , Vaiman M.
Published: August 2005

Flow cytometry analysis was applied to swine chromosomes prepared from phytohemagglutinin (PHA) stimulated peripheral blood lymphocytes. Flow karyotypes from both sexes and from t(3;7) translocation carrier females were obtained. A certain number of chromosome pairs could be assigned to various peaks. In fact, 13 peaks were observed for 18 autosomal pairs plus X and Y. Moreover, abnormalties owing to the t(3;7) translocation were readily observable.

The number of base pairs for chromosomes associated with the various peaks was estimated by comparison with human flow karyotypes.

The following four peaks were thus sorted: (1) the peak assumed to represent the translocated chromosome 7 plus the normals associated with it; (2) the corresponding peak from a normal swine; (3) the peak assumed to contain among others the normal chromosome 7; and finally (4) the peak corresponding to swine chromosome 1.

Chromosomes of each peak were collected on Pall Biodyne membrane. Following appropriate denaturation and prehybridization, the four samples were hybridized with a human leucocyte antigen (HLA) class I32P‐labelled cDNA probe, representing most of the coding sequence of the HLA B7 gene. The results confirmed previous data from other techniques that assigned the swine MHC(SLA) to chromosome 7.

Subsequently, sorted samples were hybridized with a porcine genomic Interferon alpha probe in order to confirm the mapping of this gene family on porcine chromosome 1.
Carcinoma in situ specimen classification based on intermediate cell measurements
Authors: Katzko Michael W. , Pahlplatz Martin M. M. , Oud Peter S. , Vooijs G. Peter
Published: August 2005

In this study the possibility of classifying carcinoma in situ and normal specimens by measuring normal‐appearing intermediate cells was explored. Twenty‐five histologically verified carcinoma in situ specimens and 99 normal specimens, matched with the abnormal specimens for age and use or nonuse of an oral hormonal contraceptive, were examined. The smears were monolayer preparations stained with Thionin‐Feulgen Congored. Twenty‐one nuclear features were measured. A discrimination among the experimental groups could be made on the basis of the relationship between two features, area and average optical density (AOD). A regression of AOD on area for each smear was performed. The correlation, coefficient of variation, slope, intercept, as well as the mean of the AOD level and the age of the subject were used in a discriminant analysis. This resulted in a smear classification with a false‐positive rate of 14% and a missed‐positive rate of 32%. When contraceptive use was taken into account the overall classification was improved with a false‐positive rate of 12% and a missed‐positive rate of 20%.
Comparison of tissue disaggregation techniques of transitional cell bladder carcinomas for flow cytometry and chromosomal analysis
Authors: Smeets A. W. G. B. , Pauwels R. P. E. , Beck H. L. M. , Feitz W. F. J. , Geraedts J. P. M. , Debruyne F. M. J. , Laarakkers L. , Vooijs G. P. , Ramaekers F. C. S.
Published: August 2005

DNA index (DI) measurements and chromosomal analysis of 42 transitional cell carcinomas were done after mechanical and enzymatical disaggregation of the tumor specimens. The results obtained with these different disaggregation techniques wee compared in the 33 cases (79%) that showed recognizable chromosomes. The enzymatically obtained cell suspensions could not be used for chromosomal analysis after short‐term culture of 24 hours. In four cases, the DI after enzymatical treatment could not be estimated. In most cases, the DI obtained from the tumor cells was similar for both aggregation techniques, with the exception of four cases of enzymatically treated cell suspensions in which the DI could not be estimated. The average DI of the aneuploid tumors was 13% higher than the corresponding chromosome count.

In 19% of the aneuploid tumors the proportion of aneuploid cells could not be measured after enzymatical treatment. In the remaining suspensions the proportion of diploid cells was higher after enzymatical disaggregation than after mechanical treatment. It is concluded that for flow cytometric and direct chromosomal analysis of bladder tumors, the mechanical disaggregation technique is most suitable.
Membrane fluidity measured by fluorescence polarization using an EPICS V cell sorter
Authors: Fox Michael H. , Delohery Thomas M.
Published: August 2005

We have characterized the measurement of fluorescence polarization on single cells using an EPICS V cell sorter. A critical analysis is made of the balancing and calibration of the system. The system is highly linear for polarization measurements. Cellular membranes were labeled with 1,6‐diphenyl‐1,3,5‐hexatriene (DPH) to measure membrane fluidity. Fluorescence polarization histograms had coefficients of variation as low as 7%. Cells labeled with DPH after 24 hr incubation in medium lacking serum showed a significantly higher fluorescence polarization than cells in medium containing serum. The fluorescence polarization measured at 15°C was 0.311 compared to 0.270 at 25°C for cells labeled with DPH, verifying that temperature affects the membrane fluidity as measured by flow cytometry.
Spectroscopic properties of fluorescein in living lymphocytes
Authors: Kinoshita Shuichi , Fukami Tatsuya , Ido Yutaka , Kushida Takashi
Published: August 2005

Detailed studies have been performed on various spectroscopic properties such as time dependence and excitation wavelength dependence of the fluorescence anisotropy for fluorescein molecules introduced into rat thymus lymphocytes. Experimental results have been found to be well interpreted in terms of the coexistence of two types of dye molecules, i.e., free and bound molecules. The fluorescence spectrum of only the bound molecules has been obtained from the difference in the time‐resolved spectra of fluorescence with two polarization directions. The time gate has been set at a sufficiently late time after the excitation, so that the polarization memories of the free molecules are lost. The spectrum thus determined agrees very well with that calculated from the spectral data in the stationary condition. From the above results, we come to the conclusion that the main factors which determine the fluorescence anisotropy inside the cell are the fraction and the anisotropy of the bound dye molecules. Finally, we discuss how these factors are related to biological quantities.
Flow cytometric analysis of Chlamydia trachomatis interaction with L cells
Authors: Waldman Frederic M. , Hadley W. Keith , Fulwyler Mack J. , Schachter Julius
Published: August 2005

Immunofluorescent staining and flow cytometric analysis have been investigated as means of studying the early stages of in vitro infection of Chlamydia trachomatis. The lymphogranuloma venereum strain of C. trachomatis was grown in vitro in L cells, fixed in p‐formaldehyde, stained with fluorescein isothiocyanate (FITC)‐conjugated monoclonal antibody to the chlamydial major outer membrane protein, and analyzed flow cytometrically. Infected cells stained 50–100 times more intensely than uninfected cells, and they could easily be discriminated by flow analysis. The number of infected cells and the fluorescence intensity of individual cells were proportional to the multiplicity of infection. The attachment of purified elementary bodies to L cells could be analyzed by immunofluorescence and flow cytometry. Cells exposed to 0.26 inclusion‐forming units/cell could be discriminated from an unexposed population. Flow analysis of purified elementary bodies was possible after fluorescent staining with the aid of a laser‐based cytometer and gating on low volume.
Factors governing the flow cytometric analysis and sorting of large biological particles
Authors: Harkins Kristi R. , Galbraith David W.
Published: August 2005

We have investigated the suitability of large flow cell tips for the flow cytometric analysis and sorting of large biological particles, including plant cells (pollen) and protoplasts. Using flow tips ranging in diameter from 79–204 μm, we have optimized conditions for the establishment of a stable hydrodynamic flow leading to accurate droplet production. We describe instrument modifications required for large particle sorting and demonstrate the use of these experimental conditions for the sorting to high purity of pollen and viable plant protoplasts possessing diameters as large as 95 μm. Our experiments have revealed a complex interaction among sorting efficiency, particle diameter, flow cell tip diameter and bimorphic crystal drive frequency. This interaction can be satisfactorily explained in terms of interference effects owing to phase differences between the particle‐induced disturbance and the undulation driven by the bimorphic crystal.
Flow cytometric measurements of fluorescence energy transfer using single laser excitation
Authors: Szöllösi János , Mátyus László , Trón Lajos , Balázs Margit , Ember István , Fulwyler Mack J. , Damjanovich Sándor
Published: August 2005

Flow cytometric energy transfer (FCET) measurements between labeled specific sites of cell surface elements (Szöllosi et al., Cytometry, 5:210–216, 1984) have been extended in a simplified form using a flow cytometer equipped with single excitation beam. This versatile and easily applicable method has several advantages over any nonflow cytometric (i.e., spectrofluorimetric) energy transfer measurements on cell surfaces: (1) The labeled ligands can be applied in excess, without washing, thereby enahling the investigation of relatively labile receptor‐ligand complexes. (2) Contributions of signals from cell debris, from cell aggregates, or from nonviable cells can be avoided by gating the data collection on the light scatter signal. (3) The heterogeneity of the cell population with respect to the proximity of the labeled binding sites can be studied. (4) In the cases of homologous ligands or of ligands binding to the same molecule but at different epitopes, the determination of fluorescence resonance energy transfer efficiency values can be carried out on a cell‐by‐cell basis, offering data on intramolecular conformational changes.

This modified FCET method enabled us to demonstrate the uniform density of glycoproteins, specific for Con A binding, in the plasma membrane of normal and Gross virus leukemic mouse cells of different sizes. The utility of this procedure has also been demonstrated by using the mean fluorescence intensities of the distribution curves in the calculation of the fluorescence energy transfer efficiency.
Interctions of pyronin Y(G) with nucleic acids
Authors: Kapuscinski Jan , Darzynkiewicz Zbigniew
Published: August 2005

Spectral properties of pyronin Y (PY) alone or in complexes with natural and synthetic nucleic acids of various base compositions have been studied in aqueous solution containing 10 or 150 mM NaCl and 5 mM Hepes at pH 7.0. The dimerization constant (KD = 6.27 × 103, M−1) and the absorption spectra of the dye in monomeric and dimeric form were established. The complexes of PY with single‐stranded (ss) nucleic acids show a hypsochromic shift in absorption, and their fluorescence is quenched by over 90% compared to free dye. In contrast, complexes with double‐stranded (ds) RNA or DNA (binding by intercalation) exhibit a bathochromic shift in their absorption (excitation) spectrum, and their fluorescence is correlated with the base composition of the binding site. Namely, guanine quenches fluorescence of PY by up to 90%, whereas A, C, I, T, and U bases exert a rather minor effect on the fluorescence quantum yield of the dye. The intrinsic association constant of the dye to ds RNA (Ki = 6.96 × 104, M−1) and to ds DNA (Ki = 1.74 × 104, M−1) was measured in 150 mM NaCl; the binding site size was 2‐3 base pair for both polymers. Implications of these findings for qualitative and quantitative cytochemistry of nucleic acids are discussed.
Application of pyronin Y(G) in cytochemistry of nucleic acids
Authors: Darzynkiewicz Zbigniew , Kapuscinski Jan , Traganos Frank , Crissman Harry A.
Published: August 2005

Chinese hamster ovary (CHO) cells or isolated nuclei were stained with pyronin Y (PY) and analyzed by absorption or fluorescence microscopy, as well as by flow cytometry. Specificity of the staining reaction was assayed by testing sensitivity of the stainable material to RNase or DNase. The colored complexes detected by light absorption in fixed cells stained with PY are nonfluorescent and are most likely the products of condensation of single‐stranded (ss) RNA by PY; the poly(rA) and poly(rA, rG) are the most sensitive to condensation.

The products of PY interaction with double‐stranded (ds) nucleic acids are fluorescent and can be detected in cells by cytofluorometry. PY used alone stains both DNA and RNA, and the staining capabilities of these nucleic acids vary depending upon the PY concentration at equilibrium; at a concentration above 330 μM, the RNA stainability decreases, perhaps due to its denaturation and condensation caused by the dye. In the presence of Hoechst 33342, PY can specifically stain RNA in fixed cells or isolated cell nuclei. Because only complexes of PY with ds RNA are fluorescent, this dye can be used as a probe of RNA conformation, e.g., to monitor denaturation of RNA in situ. The RNA stainability of mitotic cells is about 25% lower than that of cells in G2 phase, which indicates that during mitosis proportionately less cellular RNA is in the ds conformation. The advantages and limitations of the two cytochemical methods for DNA/RNA detection, one based on the use of Hoechst 33342 and PY, and another employing the metachromatic properties of acridine orange, are compared.
Method for detection and isolation of cholesteryl ester‐containing “foam” cells using flow cytometry
Authors: Kruth Howard S. , Cupp James E. , Khan Mushtaq A.
Published: August 2005

Accumulation of cholesteryl ester within vascular cells is a defining characteristic of atherosclerotic lesions. Therefore, it is of interest to be able to monitor this critical event in the development of atherosclerosis. With this objective in mind, we have developed a method for the detection of cholesteryl ester‐containing cells (i.e., foam cells) in cell suspensions prepared from enzymatically dissociated aortas.

Cholesteryl ester in aortic cells was selectively stained with the fluorescent dye filipin. Because filipin binds to unesterified cholesterol but not to esterified cholesterol, it was necessary first to remove unesterified cholesterol from cells by ethanol extraction so that its presence would not interfere with the specific detection of cholesteryl ester. Then unesterified cholesterol made available by enzymatic hydrolysis of cellular cholesteryl ester could be specifically stained with filipin. The filipin‐stained cell suspensions were analyzed using flow cytometry. With a flow cytometer it was possible to detect and sort cholesteryl ester‐containing cells onto glass slides for microscopic analysis.

Cell suspensions prepared from either grossly normal or atherosclerotic swine aortas contained cells with cholesteryl ester inclusions. As expected, these cells were more numerous in the atherosclerotic aortas. Cells with higher levels of fluorescence contained more numerous cholesteryl ester inclusions. Flow cytometric detection of cholesteryl ester‐containing cells should be generally useful in studies of cellular cholesterol metabolism as well as in specific studies of cellular cholesterol accumulation in atherosclerotic vessels.
In vivo cell kinetic effects of cis‐platinum on human ovarian cancer xenografts measured by dual parameter flow cytometry
Authors: Sevin Bernd‐uwe , Pollack Alan , Averette Hervy E. , Ramos Reinaldo , Donato Daniel
Published: August 2005

Dual parameter flow cytometry, measuring DNA and nuclear protein of individual cell nuclei simultaneously, makes it possible to follow cell kinetic perturbations in six distinct compartments of the cell cycle following chemotherapy in vivo. Human ovarian cancer xenografts in nude mice from a primary and recurrent tumor of the same patient were studied. The response to intraperitoneal application of cis‐platinum was assessed by tumor volume measurements, changes in labeling indices by autoradiography, and dual parameter flow cytometry. Sequential tissue samples were taken from each tumor using fine needle aspirations as a microbiopy method. Pretherapy samples were compared to multiple specimens collected up to 18 days after therapy. Morphologic changes of each specimen were also assessed. Cis‐platinum affects malignant cells in the G1B, S, G2A, and G2B compartments with various intensities and different time frames, depending on the drug sensitivity of each individual tumor.
Evaluation of contextual analysis for computer classification of cervical smears
Authors: Garcia Gerardo L. , Kuklinski Walter S. , Zahniser David J. , Oud Peter S. , Vooys Peter G. , Brenner John F.
Published: August 2005

A procedure for automated analysis of cervical smears has been implemented in an image cytometry system. Smears are described exclusively in terms of global and contextual information extracted by pattern‐recognition algorithms and represented by a vector of proportions of cellular object types. Linear discriminant functions, based on a Fisher criterion, are derived to classify smears with a cross‐section of diagnoses into two broad categories, normal and abnormal. Results obtained from 83 smears indicate 78% correct classification. In contrast to most automated systems, good classification results were obtained in normal smears with benign changes caused by inflammation and with postmenopausal atrophia and in abnormals with mild dysplasia. These findings suggest that contextual analysis may be sensitive to subtle changes in cellular morphology and to progressive patterns of dysplasia. When used with standard isolated cell analysis, contextual analysis may provide additional complementary information for automated cervical prescreening.
Display of three‐parametric data acquired by a flow cytometer
Authors: Ormerod M. G. , Payne A. W. R.
Published: August 2005

A program for producing contour maps of three‐parameter flow cytometric data is described. The program is written in Pascal and can be implemented on any microcomputer with a graphics screen.
The reliability of microspectrophotometric and flow cytometric nuclear DNA measurements in adenocarcinomas of the breast
Authors: Fallenius Anders G. , Askensten Ursula G. , Skoog Lambert K. , Auer Gert U.
Published: August 2005

The reliability of microspectrophotometric (MSP) and flow cytometric (FCM) nuclear DNA measurements has been studied in 50 human breast adenocarcinomas. The tumor material was obtained by means of fine‐needle aspiration biopsy, and all samples except one were found to be highly representative. The results confirm earlier observations that a good correlation exists between modal value (MV) determined by MSP and DNA index (DI) determined by FCM. However, when tumors were classified into low and high malignant variants according to FCM/DI, FCM/S‐phase percentages, and MSP histogram types, the concordance was less pronounced. This was found to be due mainly to the fact that in near‐diploid tumors a discrepancy exists between MSP and FCM ploidy, as well as between MSP distribution pattern and the estimated percentages of cells in the S‐phase region. Another source of discrepancy was observed in tumors with stemlines in the normal tetraploid region, including cells with highly scattered aneuploid DNA values. These tumors were judged by MSP as aneuploid/high malignant and by FCM as euploid/low malignant. In view of this discrepancy, we conclude that the simple determination of the stemline position by MSP/MV or FCM/DI is not sufficient for adequate cytochemical malignancy grading of breast carcinomas. We suggest that a combination of ploidy and percentage of cells scattered outside the modal peaks is a more sensitive method for optimal cytochemical malignancy grading in breast carcinomas.
Improved model for specimen classification based on single‐cell classifiers
Authors: Cox C. , Wheeless L. L. , Reeder J. E. , Robinson R. D. , Berkan T. K.
Published: August 2005

We consider probabilistic models for specimen classification procedures based on systems which classify individual cells as normal or abnormal. The models which we consider generalize those discussed previously by Castleman and White (Anal. Quant. Cytol. 2:117–122, 1980; Cytometry 2:155–158, 1981) and by Timmers and Gelsema (Cytometry 6:22–25, 1985). In particular, they include the biologically plausible possibility that the specimen contains cells which are intermediate between the extremes of normal and abnormal. We find that if these additional cells occur differentially in normal and abnormal specimens, then specimen classification can become substantially more efficient when the cell classifier has different error rates for these cells.