bluuebunny/crossref_metadata_2025 huggingface dataset:
Design and operation of a dual laser chromosome sorter
Authors: Lebo Roger V. , Bastian Araxy M.
Published: August 2005

We describe the design and operation of a dual laser fluorescence activated chromosome sorter that can analyze and sort chromosome suspensions stained with two complementary DNA stains. We used two 18‐watt argon‐ion lasers to provide maximal excitation of the DNA‐specific stains Hoechst 33258 and chromomycin A3. The lasers are focused onto the sample stream by independent optics permitting optimal focusing of any pair of laser wavelengths. We incorporated beam expanders to decrease the energy density on the optics and improve the focal spot uniformity. Adjustable focusing lens holders are used to set the optimal focal spot size. The redesigned sorter separates suspensions of normal human fibroblast chromosomes into 21 fractions and can reliably sort more pure chromosome fractions than was previously possible.
Simple methods to determine and compare the sensitivity of flow cytometers
Authors: Pinkel D. , Steen H. B.
Published: August 2005

Knowledge of the sensitivity of a flow cytometer is important both in choosing an instrument for a particular application and in determining whether the resolution obtained on real samples is limited by instrumental factors or sample variability. The coefficient of variation (CV) obtained for a measurement is given by CV2 = CVS2 + CVI2 + 1/n + K(δVB2)/n2, where CVS is the variability due to the sample, CVI that due to variations in illumination of the sample particles, n the number of photoelectrons emitted from the photomultiplier photo cathode per sample particle (which is the signal intensity), δVB2 the mean square background noise voltage (usually due to background illumination), and K a constant. Measurement of the CV of light flashes from a light emitting diode as a function of their intensity allows development of standard curves which can be used to directly determine the degree to which the resolution obtained on actual samples is limited by signal intensity and background light. The use of standard particles allows relative calibration of the curves for different instruments and which also ranks their sensitivities.
Multistation multiparameter flow cytometry: A critical review and rationale
Authors: Shapiro Howard M.
Published: August 2005

The capacity for fluorescence excitation by beams of different wavelengths at separate points along the sample stream, and the capacity for computer analysis of multiparameter data thus obtained, are now available in flow cytometer/sorter systems from commercial producers. It is now readily apparent to most experienced users of flow cytometers that such multiparameter analysis offers the most convenient solution to the problem of characterizing subpopulations of cells within a mixed population. The use of multiple beams facilitates resolution of fluorescence signals from several probes within or upon a single cell and widens the range of analytical alternatives available to experimenters. This critical review discusses the history of the instrumentation, the parameters now measurable and the probes used for their measurement, and the methods for data analysis. Required sensitivity and precision are discussed, leading to the conclusion that many of the advantages of multistation, multiparameter flow cytometry can be made available in less complex and less costly instruments using less powerful sources and less elaborate computer hardware than are presently incorporated in commercial apparatus.
Application of the CYTOMIC 12 flow cytometric compact analyzer for automatic kinetic measurements
Authors: Kachel V. , Schneider H. , Bauer J. , Malin‐Berdel J.
Published: August 2005

Flow cytometry, usually applied to cells which have time independent features, can also be used for kinetic experiments where the change of cell populations with time is investigated. Dedicated time sequencing programs written in Assembler and incorporated in the CYTOMIC 12 analyzer (4) are described. A sequence of 64 one parameter histograms can be automatically acquired and immediately displayed as a pseudo‐two‐parameter histogram. The acquisition time for each of the subsequent histograms can be selected between 1 and 32 seconds. Kinetics lasting up to 34 minutes are resolved into 64 time intervals. Two parameter kinetics can be resolved into 12 32 × 32 channel, two parameter histograms which are displayed and evaluated immediately on the analyzer screen in groups of 4 without using complicated list mode procedures. The standard CYTOMIC 12 software can be applied for processing and printing of the sequence distribution curves.
Nondiscrete heterogeneity of human erythrocytes: Comparison of coulter‐principle flow cytometry and Soret‐hemoglobinometry image analysis
Authors: Bessman J. David , Hurley Edward L. , Groves Michael R.
Published: August 2005

In the blood of normal subjects, the volumes of single erythrocytes are distributed with a coefficient of variation (cv) of 10.8 ± 1.8%; while in hemoglobinopathies, cv increases proportionately to the degree of anemia produced. Using single cell Soret‐band hemoglobinometry and focused‐aperture impedance counting, we compared the distribution of red cell volume, area, hemoglobin content, and hemoglobin concentration in normals and subjects with anemic disorders. The cv, nondiscrete heterogeneity, is first, a general characteristic of biologic measurement, second, a sensitive indicator of abnormality of erythropoiesis, and third, consistently less for hemoglobin concentration than for volume, area, or hemoglobin content of the same cells.
Behavior of elliptocytes under shear stress in the rheoscope and ektacytometer
Authors: Bull Brian , Feo Claude , Bessis Marcel
Published: August 2005

When ellipsoidal red cells from patients with hereditary elliptocytosis are subjected to shear stress in a rheoscope the cells rotate around their short axis and align themselves perpendicular to the flow. At high shear stress they deform into an ellipsoid of revolution with the major axis of the ellipse normal to the flow. This change in the direction of the long axis is not the result of simple reorientation of the cells. Rather, the short axis of an elliptocyte exposed to increasing shear stress expands while its long axis contracts. It is this process that eventually brings about the apparent reorientation.

Elliptocytes in a well mixed suspension are randomly oriented. As a consequence, the initial diffraction pattern displayed by the ektacytometer is a hazy circle. As the shear stress is increased this becomes rhomboid due to expansion of the central region of each cell (a change confirmed by the rheoscope). Central expansion coexisting with polar remnants of the resting elliptocytic cell shape gives rise to the rhomboid outline. As the shear stress in the ektacytometer approaches 120 dynes/cm2 the characteristic, elliptical diffraction pattern seen with normal cells makes its appearance. As the shear stress is decreased, the rhomboid pattern reappears followed by a new elliptical pattern oriented at right angles to the one seen under high shear. This pattern arises from the resting shape of the elliptocytes which have all been oriented at right angles to the shear stress as it decreased to zero. As a consequence of this uniform cellular orientation, all exposures to shear stress after the initial one start with a deformability index of less than zero if it is calculated in the usual fashion from the axial ratio of the shear deformed cells. Certainly under these circumstances and possibly in other conditions as well the use of the less‐specific term “ektacytometric index” would be less misleading.
Setting up and running a microscope‐based flow cytometer
Authors: Petersen Steffen E.
Published: August 2005

A noncommercial flow cytometry system was set up in a laboratory with no engineering staff attached. The system is based upon a conventional fluorescence microscope and a new flow principle reported by H. Steen and T. Lindmo (Science 204:403, 1979). A few modifications and additions have been introduced. The instrument has proven to be a high‐resolution system, easy to adjust and control, giving coefficients of variation of cellular DNA down to 0.8% and of fluorescent polyethylene microspheres down to 0.9%.
Long‐term storage of samples for flow cytometric DNA analysis
Authors: Vindeløv Lars L. , Christensen Ib J. , Keiding Niels , Spang‐Thomsen Mogens , Nissen Nis I.
Published: August 2005

A simple procedure for long‐term storage of cells for flow cytometric DNA analysis was developed and tested. The cells were stored as single cells or fine‐needle aspirates suspended in a citrate buffer with dimethylsulfoxide (DMSO), or as small blocks of tissue from solid tumors. The cells were stored for up to one year by freezing at —80°C. Statistical analysis of the results showed no change in the fractions of cells in the cell cycle phases as determined by deconvolution of the DNA‐histograms. It was found that in addition to the intrinsic sample variation from the parameter estimation by deconvolution, there was significant intraday and interday variation. Hence the most accurate results are obtained if different aliquots of a sample are measured on different days rather than on the same day. Use of the storage method thus has the potential of increasing the accuracy of the analysis. The storage method makes sample collection independent of immediate subsequent analysis. This has enabled us to perform large internally controled experiments, involving more samples than can be analyzed in one day, to examine tumor sampled from different hospitals and to utilize fully the capacity of our flow cytometer. The method was a prerequisite for developing an accurate standardization procedure for DNA content determination.
A Detergent‐trypsin method for the preparation of nuclei for flow cytometric DNA analysis
Authors: Vindeløv Lars L. , Christensen Ib J. , Nissen Nis I.
Published: August 2005

A new modification of our detergent technique for the preparation of nuclei for flow cytometric DNA analysis is described. The attainment of low coefficients of variation of the peaks and of quantitative staining of nuclei from different tissues was a problem with the original method. This was solved in the new modification by trypsinization of the unfixed nuclei. The nuclei were stabilized by spermine. A simple procedure for long‐term storage of samples at —80°C was integrated into the method. The fluorescence of the nuclei was stable for at least 3 hours after staining. Light exposure protection of the samples was essential. No cell loss was caused by storage or staining. The method was successfully applied on samples including: (a) Normal tissues— human lymphocytes, granulocytes and spleen. Mouse lymphocytes, bone marrow, spleen, liver, kidney and thymus. (b) Human neoplasms— lung cancer, breast cancer, lymphoma, leukemia, bladder cancer and cancer of the oral cavity. (c) Human tumors in nude mice— breast cancer, lung cancer, melanoma and colon cancer. (d) Mouse ascites tumors— JB‐1, L 1210, Ehrlich and P 383. It therefore seems well suited as a routine clinical procedure.
Standardization of high‐resolution flow cytometric DNA analysis by the simultaneous use of chicken and trout red blood cells as internal reference standards
Authors: Vindeløv Lars L. , Christensen Ib J. , Nissen Nis I.
Published: August 2005

Determination of nuclear DNA content by flow cytometry requires comparison with a reference standard. The use of external standards such as lymphocytes or granulocytes is time‐consuming and inaccurate. Chicken red blood cells (CRBC) have a DNA content of 35% of the human diploid value and have been widely used as internal standard. The ratio calculated on the basis of the peak channel numbers of the standard and the sample and used to indicate the DNA content (DNA ratio) is, however, very sensitive to changes in the zero level adjustment of the flow cytometer. If two internal standards are used the DNA ratio becomes independent of the zero level. Rainbow trout red blood cells (TRBC) have a DNA content of 80% of human diploid cells. A mixture of CRBC and TRBC was prepared and stored in small aliquots at —80°C. This mixture was added to the sample before staining. The day‐to‐day variation of the DNA ratio obtained by use of the two standards was smaller than that obtained by CRBC alone. The possibility of sex related differences in DNA content of CRBC and TRBC was examined. The results indicated that a new batch of standards should be tested against the old batch to avoid the introduction of a systematic error.
Limits of detection of nuclear DNA abnormalities by flow cytometric DNA analysis. Results obtained by a set of methods for sample‐storage, staining and internal standardization
Authors: Vindeløv Lars L. , Christensen Ib J. , Jensen Grethe , Nissen Nis I.
Published: August 2005

Flow cytometric DNA analysis is an attractive alternative to conventional cytogenetics for diagnosing karyotype changes resulting in abnormal DNA content. We have developed methods for long‐term storage of samples and standards, for staining the cells and for standardization of the measurements by two internal standards. In this paper the currently attainable resolution was determined and limiting factors were identified. Normal reference values for male and female leucocytes were determined by analyzing 240 samples from six men and six women, all cytogenetically normal. The DNA content of female cells was 1.5% higher than that of male cells. This finding was used to correct the results for sex related DNA differences. The 95% confidence limits for mononuclear blood cells were ±0.79%. The results on granulocytes were exceptionally variable, with 95% confidence limits of ±1.79%. Reexamination of the normal leucocytes after 1 year showed a long‐term drift of up to 2% in the results, indicating a need for regular checks of the reference values. In addition to the sex related differences in DNA content, individual differences of up to 1% were demonstrated. This creates problems as to which reference value to use for a particular sample. Furthermore, tissue related differences in fluorescence were found when six different tissues from one mouse were examined. The means had a range of 0.7%. The tissue related differences add uncertainty to the interpretation of the results. The resolution of heterogeneous populations with slightly different DNA content was examined by analyzing mixtures of cells with known DNA differences ranging from 1.50% to 5.83%. With a coefficient of variation of the peaks of about 2% a DNA difference of approximately 4% was required for accurate determination of the DNA content of the two individual populations. A lower coefficient of variation would increase the resolution, but tissue related differences in fluorescence could then become the limiting factor.
Characterization of the uptake and toxicity of a fluorescent thiol reagent
Authors: Olive Peggy L. , Biaglow John E. , Varnes Marie E. , Durand Ralph E.
Published: August 2005

3‐(4‐Maleimidylphenyl)‐4‐methyl‐7‐diethylamino coumarin (CPM), is a fluorescent thiol‐binding agent. CPM is nontoxic to Chinese hamster V‐79 cells (2 × 105 cells/milliliter) exposed to 2.5 μg/milliliter for 30 minutes. However, both toxicity and cellular binding were directly dependent on the drug:cell ratio. Using flow cytometry, cellular binding of CPM correlated with inhibition of DNA synthesis. In cells pretreated with the thiol binding drugs N‐ethylmaleimide, diamide and diethylmaleate or the carcinogens 4‐nitroquinoline‐1‐oxide and AF‐2 (furylfuramide), the subsequent binding of CPM was reduced by 40% or more.
Optimization of mammalian chromosome suspension preparations employed in a flow cytometric analysis
Authors: Th. Bijman J.
Published: August 2005

Metaphase chromosomes from Chinese hamster V‐79 cells have been stained with propidium iodide and their DNA content was measured by flow cytometry. The chromosomes in suspension must be stabilized during the preparation to preserve their morphological integrity. In order to obtain high resolution in the flow karyotype, several modifications were made to the method used to prepare chromosome suspensions. The influence of several ions, detergents and detergent mixtures was investigated. Magnesium (3‐5 mM) together with sodium (10 mM), Tris (10 mM) and Triton X‐100 (0.4%) gave the best results. In contrast, sodium and potassium exerted no effect, while calcium had a negative effect on the preparation as measured with flow cytometry. Compared with a solution of potassium chloride + propidium iodide, chromosomes prepared in Tris buffer resolved better with less background. A histogram quality factor which contains the variables peak height, depth of valley and coefficient of variation, was used to quantitate the histograms.
High resolution method for the analysis of DNA histograms that is suitable for the detection of multiple aneuploid G1 peaks in clinical samples
Authors: Schuette W. H. , Shackney S. E. , Maccollum M. A. , Smith C. A.
Published: August 2005

The DNA histogram obtained by flow cytometry can be considered as the product of an “ideal” measurement column vector and a measurement distortion matrix. In order to extract the ideal histogram from the real data, the measurement distortion matrix is commonly presumed to be a family of Gaussian coefficients that are centered on the diagonal. We have designed a feedback‐controlled curve‐fitting procedure that reconstructs the ideal histogram from the real data through successive iterations. The optimum coefficient of variation (cv) for the family of Gaussians in the measurement distortion matrix is determined from an analysis of the sums of squares of fits of the computed DNA histogram to the real data over an appropriate range of trial cv. Since this method assigns a Gaussian to each and every data channel, it permits the resolution of closely spaced multiple aneuploid G1 peaks in clinical samples. The effects of high frequency noise that may be present in the data can be attenuated by multiplying the real data histogram by a Gaussian matrix with cv close to but smaller than that of the measurement distortion matrix.
DNA flow cytometric analysis of monogranulocytic colony forming cells from mouse bone marrow in Vitro
Authors: Ronot X. , Adolphe M. , Hecquet C. , Fontagne J. , Jaffray P. , Lechat P.
Published: August 2005

An adaptation of a previously reported flow cytometric technique is described. This technique was applied to study the DNA distribution of mouse granulocyte‐macrophage colony forming cells (GM‐CFc) grown in a methyl cellulose culture system. This method involved the collection of cell clusters and colonies from the methyl cellulose culture medium for the subsequent determination of DNA content. The DNA content of the homogeneous GM‐CFc suspensions was determined by applying a modification of the propidium iodide staining procedure described by Crissman et al. The relative percentage of cells in G0 + G1, S, and G2 + M stages of the cell cycle was calculated by applying mathematical analysis to the resulting DNA histograms. This method could be useful in studying the effects of drugs on GM‐CFc kinetics.
A simple density gradient for enriching subfractions of solid tumor cells
Authors: Walle Alexander , Kodama Tetsuro , Melamed Myron R.
Published: August 2005

Cell suspensions of human solid tumors could be separated into 5 or 6 subfractions for flow cytometry by centrifugation on a continuous silica gel (Percoll, Pharmacia Fine Chemicals, Upsalla, Sweden) density gradient. In all cases, there were one or more subfractions with enrichment of the aneuploid tumor cells. The greatest enrichment occurred in cases with fewer than 20% aneuploid cells in the original specimen; for these cases the enrichment factor ranged from 2.8‐ to 11.7‐fold increase. In six cases with fewer than 7% aneuploid G0/1 cells initially, the aneuploid G0/1 DNA peak was difficult to demonstrate on the original specimen but became quite obvious in the subfraction. In no case were the tumor cells confined to a single subfraction. The distribution of tumor cells within two or more subfractions appeared related to differentiation in the case of squamous cells and to RNA content for other tumor cells. DNA/RNA flow cytometry proved useful in defining subpopulations of tumor cells with different DNA stemlines and varying positions in the cell cycle according to the RNA content of the respective subpopulation.
Classification of immature and mature cells of the neutrophil series using morphometrical parameters
Authors: Bins M. , van Montfort L. H. , Timmers T. , Landeweerd G. H. , Gelsema E. S. , Halie M. R.
Published: August 2005

Quantitative morphological data of six classes of immature and mature cells of the neutrophil series of the bone marrow of normal persons were used for statistical classification experiments (myeloblasts, promyelocytes, myelocytes, metamyelocytes, bands and segments). On each cell, parameters were measured directly from the image or calculated from the shape of the density histogram or the counting densitogram using a Texture Analysis System (E. Leitz, Wetzlar, Germany). The parameters were analyzed with the interactive statistical pattern recognition system ISPAHAN. One half of the data were used as a learning set and the other half as the test set. The parameters were compared according to their performance in discrimination between the classes, alone and in combinations. Parameters not contributing to an improvement of the discrimination were disregarded. Eleven parameters were selected and used for classification by two different methods: a stepwise and a “one‐shot” method. Stepwise classification resulted in a 79% correct classification rate. Most errors occurred between cell classes in neighboring stages of maturation. In 96% of all cases the computer classification was either in accordance with that of the technician or with a cell class of a neighboring maturation stage. One step classification by the computer was in agreement with the technicians in 82% of the cases. For 98% of the cells the computer classification was either in accordance with that of the technician or with a cell class of a neighboring maturation stage.

The data set was collected by two technicians, operating independently. Differences in their interpretation of the maturation stage were found by comparing the performance of classifiers based on both cell samples. Since the images of the cells were not available for reexamination, the causes of disagreement in classification between the technicians and between computer and technicians could not be evaluated.
A flow cytometric study of the propidium iodide staining kinetics of human leukocytes and its relationship with chromatin structure
Authors: Mazzini Giuliano , Giordano Paolo , Riccardi Alberto , Montecucco Carlo Maurizio
Published: August 2005

A nonstoichiometric staining method based on the employment of propidium iodide at a concentration far below saturation is described. The method is particularly suitable for the measurement of staining kinetics and it allowed us to differentiate cell populations with the same DNA content but different chromatin conformation. Analyses have been performed on normal and pathologic human leukocytes. Normal mononuclear cells have been differentiated from polymorphonuclear cells and lymphoblasts.
DNA cytometry on four‐micrometer sections of paraffin‐embedded human renal adenocarcinomas and adenomas
Authors: Bennington James L. , Mayall Brian H.
Published: August 2005

This study assessed the diagnostic and prognostic utility of DNA cytometry on 4 μm sections of paraffin‐embedded human renal adenocarcinomas and adenomas.

Twenty‐one renal cortical glandular neoplasms from patients who had been followed for up to 12 years after surgical removal of their tumors were examined. These tumors were classified histopathologically and included six adenomas and fifteen adenocarcinomas (six grade I, five grade II, three grade III, and one grade IV). For each tumor, 4‐μm sections were cut from paraffin‐embedded blocks retained from the original surgical specimen. Both tumor and normal renal cortex of the same kidney were included on each slide and stained with the azure A Feulgen reaction for DNA. Nuclei from 200 tumor cells and 100 control proximal convoluted tubular cells were selected at random for measurement of nuclear DNA stain content utilizing an integrating image cytometer. For each tumor, the cytometric measurements were standardized to the corresponding control measurements, plotted as histograms, and analyzed statistically; the distributions of the standardized measurements were described by features such as mean, variance, and interquartile range.

The locations and shapes of the tumor histograms differed markedly from their controls. The values of the features all were significantly greater for each tumor than for its normal tissue control; the differences increased with increasing tumor grade. Multiple regression analysis of the features against histopathologic grade showed a clear and statistically significant correlation. This correlation allowed accurate prediction of tumor grade from the cytometric data. The cytometric features also discriminated accurately between patients with favorable and unfavorable outcomes. However, they showed no clear distinction between renal adenomas and grade I adenocarcinomas nor between oncocytic and clear cell tumors of the same grade. This study indicates that quantitative information obtained by DNA cytometry of thin sections can form the basis for improved histopathologic diagnosis and prediction of clinical outcomes for patients with renal adenocarcinoma.
Parametric analysis of histograms measured in flow cytometry
Authors: Mann R. C. , Hand R. E. , Braslawsky G. R.
Published: August 2005

Flow cytometric histograms frequently consist of several components that show various degrees of overlap. For many types of analysis it is of great importance to decompose the original histogram into its components. To that purpose, we investigated the maximum likelihood approach in detail. It is shown that the iterative method to solve the maximum likelihood equations is well behaved for a variety of initial values. Algorithms to obtain initial values are presented, and the performance of the method is tested when applied to the analysis of DNA measurements from heterogeneous cell populations that differ with respect to DNA content.
Rapid identification and detection of parasitized human red cells by automated flow cytometry
Authors: Whaun J. M. , Rittershaus C. , Ip S. H. C.
Published: August 2005

Rapid and reliable identification of various human red cell parasites is important in many chemotherapeutic and immunologic studies. Because manual microscopic counting is tedious and imprecise, we have developed a simple diagnostic procedure for the automated flow cytometric detection of in vitro infected red cells, using a nucleic acid‐binding fluorescent dye, acridine orange. Human malaria (Plasmodium falciparum)‐infected red cells from continuous human erythrocyte culture were incubated at room temperature in acridine orange stain for 5 min after which the sample were analyzed by flow cytometry. Since mature red cells contain no DNA, infected red cells were identified with a distinct fluorescent signal. A total of 200,000 cells per sample were counted and analyzed in less than 2 min. Rings, trophozites, and schizonts were assessed and identified in synchronozed infected red cell cultures by flow cytometry. In addition, various stages of infected red cells were isolated with a cell sorter. This rapid method permits accurate and reliable assessment of data with the exclusion of anomalous data such as damaged cells, extraneous material, and contaminating particles.
Isopycnic density‐gradient centrifugation: A separation parameter which improves flow cytometric measurements on heterogeneous tumors
Authors: Šonka J. , Stöhr M. , Vogt‐Schaden M. , Volm M.
Published: August 2005

The analytic and prognostic value of flow cytometric measurements can generally be improved by preseparation of cells. The pre sent data demonstrate clearly the distinct character of bands obtained in density‐gradient separation. Density‐gradient centrifugation in Percol after extensive Ca2+ + Mg2+ elimination helps to resolve more detail in flow cytometric measurements (e.g., additional DNA stem lines in human tumors) and can be used studies of tumor heterogeneity. The significance of investigations of tumor heterogeneity is demonstrated by the different reactivity of subpopulations of the experimental murine tumor S 180 to vincristine.
Characterization of ploidy level in bladder tumors and selected site specimens by flow cytometry
Authors: Jakobsen Anders , Mommsen Søren , Olsen Steen
Published: August 2005

The histopathological grade of atypia was compared with the ploidy level in biopsy specimens from bladder tumors and six grossly normal areas (selected sites). The study included investigation of 303 specimens from 45 patients. The results demonstrated a high correlation between the grade of atypia and the occurrence of aneuploid cell populations. The selected site specimens were diploid in patients with low grade malignant tumors, while high grade malignant tumors were associated with aneuploid selected site specimens in 20% of the cases. The divergence of ploidy level in the tumor and selected site specimens found in some cases clearly demonstrated that the carcinogenic process in the bladder may lead to clonal heterogeneity.

Flow cytometric analysis seems suited for mapping of the ploidy levels in different parts of the bladder. The possible importance of the additional information is discussed.
Do all daughter cells enter the “indeterminate” (“A”) state of the cell cycle? Analysis of stathmokinetic experiments on L1210 cells
Authors: Kimmel Marek , Traganos Frank , Darzynkiewicz Zbigniew
Published: August 2005

The results of ten different stathmokinetic experiments on L1210 cells are analyzed to determine whether all the postmitotic cells enter the exponential AG1 compartment of the cell cycle characterized by exponentially distributed transit times. The analysis is based on a mathematical model coherent with the generalized A‐B‐transition hypothesis. An original fitting procedure is introduced to estimate the fraction of cells entering AG1 as well as other parameters of the cell cycle. Results of the analysis suggest that either all or nearly all postmitotic cells enter G1A. The results are discussed with respect to the validity of the A‐B‐transition hypothesis of the “probabilistic” model of the cell cycle. Some systematically occurring discrepancies that do not conform with generally accepted cell cycle models are apparent from the analysis of this data.
A method for staining 3T3 cell nuclei with propidium iodide in hypotonic solution
Authors: Tate Emily H. , Wilder Mark E. , Cram L. Scott , Wharton Walker
Published: August 2005

A modification of the propidium‐iodide hypotonic sodium citrate method has been developed specifically for high‐resolution staining of mouse 3T3 cell nuclei for analysis by flow cytometry. The method employs a brief treatment of cells at 37°C with Triton X‐100 and RNAse in the presence of propidium iodide in hypotonic sodium citrate, followed by restoration to isotonicity with NaCl. The average CV obtained for the G1 peak was 3.5%, and the samples were stable for 1–2 weeks at 4°C. Compared to this technique, previously described propidium iodide‐staining methods gave poor resolution with 3T3 cells.
Flow cytometric DNA content in myelodysplastic syndromes
Authors: Montecucco Carlomaurizio , Riccardi Alberto , Traversi Egidio , Danova Marco , Ucci Giovanni , Mazzini Giuliano , Giordano Paolo
Published: August 2005

DNA flow cytometric analysis of unfixed bone marrow cells stained with propidium iodide was carried out in 33 patients with untreated primary myelodysplastic syndromes. Patients with stable clinical course for up to 3 years had higher fractions of cells in S and G2 phases (22.7 ± 12.4% and 12 ± 3.6%) than those who developed acute leukemia and/or died early in the course of disease (14.4 ± 8.5% and 6.6 ± 4%). Median survival was more than 36 mo in patients with S+G2 cell fraction higher than 24%, and 14 mo in the remaining 16 patients with lower values (P < 0.01). Analyses repeated after 3–24 mo showed no major changes in cell proliferation pattern in ten out of 11 patients. The remaining patient had sharp decrease in S and G2 cell fraction 3 mo before the transition into acute leukemia. The DNA index (DI) of bone marrow cells was calculated to assess ploidy. However, comparative evaluation of cytologic, cytogenetic, and flow cytometric data suggest that, under our experimental conditions, the DI may be influenced by factors such as the degree of chromatin compactness.
Continuous measurement and analysis of staining kinetics by flow cytometry
Authors: Beumer Tom , Pennings Arie , Beck Hans , Wessels Johan , Haanen Clemens , Herman Chester
Published: August 2005

The measurement of color development with time in cells following the start of a staining reaction is of interest in a number of biological systems. These include the subsets of peripheral white blood cells after acridine orange staining, the uptake by cells and nuclei of fluorescent agents, especially antitumor drugs, and measurement of intracellular enzyme kinetics using fluorogenic or absorbing substrates. The present work describes a simple computer program for analyzing flow cytometric (FCM) data versus time, including both the population kinetics of color development and the variability of staining speed within one population of cells.

A single‐channel absorption measurement in flow (Technicon Hemalog D) was used to record peroxidase kinetics in peripheral blood cells. Every 5 s, a 64‐channel absorption histogram was recorded, up to a maximum of 64 histograms. The data were then analyzed by a computer program which searched for the peak channel of each histogram. A least‐squares fit was computed for these maxima. The asymmetries of the 64 absorption histograms were compared to see if there was more than one population present with different time constants.

Although developed for enzyme kinetic measurements, this program may have wider usefulness in any measurements of time‐dependent phenomena by FCM.
Characterization and sorting of mouse cytotoxic macrophages by their light scattering properties
Authors: Martin A. , Merdrignac G. , Collet B. , Genetet B.
Published: August 2005

The light scattering properties of mouse activated macrophages were analyzed by flow cytometry. Peritoneal adherent cells from B. abortus treated animals were found to segregate into two subpopulations as a function of their foward angle and 90° angle light scatter. The cell subpopulations were separated by automatic sorting. The strongly scattering ones contained an elevated proportion of large volume and acid phosphatase rich cells. Their nonspecific cytotoxic activity against tumor cells was more important than that of weakly light scattering cells. Thus, flow cytometry might be helpful to characterize and isolate cytotoxic macrophage populations.
Simultaneous measurement of phagocytosis and phagosomal pH by flow cytometry: Role of polymorphonuclear neutrophilic leukocyte granules in phagosome acidification
Authors: Bassøe C.‐F. , Laerum O. D. , Glette J. , Hopen G. , Haneberg B. , Solberg C. O.
Published: August 2005

Human polymorphonuclear neutrophilic leukocytes (PMNLs) phagocytosed fluorescein‐isothiocyanate (FITC)‐labelled Staphy‐lococcus aureus. Free bacteria, phagocytes, and nonphagocytes were discriminated and quantified by flow cytometry (FCM). The relative fluorescence of phagocyte‐associated and free bacteria (Nf:N) was calculated by dividing the mean phagocyte fluorescence by that of the free bacteria and the number of phagocytosed bacteria. Bactericidal capacity and chemiluminescence were measured by standard methods. The red‐to‐green fluorescence ratio of acridine orange‐stained PMNLs (R/G) was measured by FCM. Degradation of bacteria was monitored by the reduction in FITC and ethidiumbromide fluorescence of bacteria liberated from the phagocytes. Bacterial FITC fluorscence was pH dependent. Nf:N was 0.5 to 0.7. Using a standard curve for the interrelationship between bacterial fluorescence and pH, phagosomal pH was 5.0–5.5 Phagocytes, kept at 4°C for 24 h had Nf:N ∼ 1, did not degrade bacteria, but killed them and emitted chemiluminescence. NH4Cl increased phagocyte fluorescence by 27% and decreased R/G by 50%. Cyanide and azide did not affect Nf:N. Nf:N of phagocytes from a patient with chronic granulomatous disease was 32% below, and R/G was 32% higher than the controls. Acidification of the phagosomes seems to be related to discharge of PMNL granule contents and independent of the respiratory burst.
Immunofluorescence measurement in a flow cytometer using low‐power helium—neon laser excitation
Authors: Shapiro Howard M. , Glazer Alexander N. , Christenson Lisa , Williams John M. , Strom Terry B.
Published: August 2005

Helium‐neon lasers are economical and efficient light sources; their utility in flow cytometry to date has been limited by the lack of fluorescent probes that can be excited at 633 nm. Allophycocyanin (APC), a highly fluorescent phycobiliprotein, can be used as an antibody label and has spectral characteristics suitable for use with He‐Ne lasers; we undertook to resolve whether a low‐power (7 mW) He‐Ne laser could provide sufficient excitation to permit flow cytometric detection of APC‐labeled antibodies on cell surfaces. We made an APC conjugate of monoclonal antibody 4F2, which reacts with an antigen abundant on the surfaces of activated human T‐lymphocytes; APC‐4F2 was used to stain blood mononuclear cells that had been cultured with and without phytohemagglutinin (PHA). Cells so stained were examined in a flow cytometer with orthogonal illumination at 633 nm from a 7 mW He‐Ne laser; antibody‐bearing cells were detectable by fluorescence emission above 665 nm. Cells from the same cultures were stained with fluorescein‐labeled 4F2 antibody and examined in a flow cytometer with argon ion laser excitation at 488 nm. Percentages of antibody‐bearing cells determined from APC fluorescence and from fluorescein fluorescence were in good agreement. It thus appears that He‐Ne lasers and APC‐antibodies are usable for immunofluorescence measurements; the sensitivity attainable with this technique remains to be determined.
Separation and analysis of human chromosomes by combined velocity sedimentation and flow sorting applying single‐ and dual‐laser flow cytometry
Authors: Collard J. G. , Philippus E. , Tulp A. , Lebo R. V. , Gray J. W.
Published: August 2005

Human chromosomes were separated on basis of size by velocity sedimentation at 52g in a specially designed sedimentation chamber. The chamber has been constructed in such a way that large numbers of chromosomes can be fractionated on a sucrose gradient while wall sedimentation, streaming, and swirling movements of the gradient during centrifugation are eliminated. Flow deflectors in the chamber allow undisturbed introduction and fractionation of the density gradient. The different chromosomal fractions obtained are highly enriched for the various human chromosomes. Individual chromosomes were subsequently sorted to purity by fluorescence activated flow sorting using a FACS IV flow sorter equipped with a 4‐W argon‐ion laser. Following this procedure, the sorting rate for specific chromosomes can be speeded up by a factor of 5–10 when the pre‐enriched chromosomal fractions are used as starting material. A high chromosome resolution could be obtained by a few simple modifications of the FACS IV. By applying fluorochromes with different DNA‐base specificity the sorting possibilities of individual human chromosomes can be improved. In addition, the chromosomal fractions were analysed by dual laser flow cytometry after staining the chromosomes with Hoechst 33258 and chromomycin A3. In this way the enrichment of virtually all individual human chromosomes in the different chromosomal fractions can be visualized.
Bivariate flout cytometric analysis of murine intestinal epithelial cells for cytokinetic studies
Authors: Pallavicini M. G. , Ng C. R. , Gray J. W.
Published: August 2005

The heterogeneous nature of the small intestine and the lack of methods to obtain pure crypt populations has, in the past, limited the application of standard flow cytometric analysis for cytokinetic studies of the proliferating crypts. We describe a flow cytometric technique to discriminate crypt and villus cells in an epithelial cell suspension on the basis of cell length, and to measure the DNA content of the discriminated subpopulations. Our data indicate that bivariate analysis of a mixed epithelial cell suspension can be used to distinguish mature villus cells, G1 crypt cells, and S‐phase crypt cells. In addition, continuous labeling studies suggest that the position of a cell on the cell length axis reflects epithelial cell maturity. We applied this flow cytometric technique to determine the cvtokinetic nature of epithelial cells obtained by sequential digestion of the small intestine.
A Computer algorithm for the analysis of protein distribution in budding yeast
Authors: Martegani Enzo , Vanoni Marco , Delia Domenico
Published: August 2005

Flow cytometry gives relevant data on cellular parameters such as DNA, RNA, and protein contents of individual cells and is therefore a powerful tool for analyzing microbial population dynamics. Relevant information about growth dynamics may be obtained from protein distribution. In fact, protein distribution is related to age distribution and depends on the law of growth of the population and the law of growth of the single cell. To extract the available information from protein distribution, we developed a computeralgorithm starting from a model for growth of Saccharomyces cerevisiae. This algorithm quantitatively fits experimental protein distributions, allows a deconvolution of these distributions, and thus yields information about temporal parameters of the cell cycle and structure of yeast populations.
Processing of staphylococcus aureus and zymosan particles by human leukocytes measured by flow cytometry
Authors: Bassϕe Carl‐Fredrik
Published: August 2005

Staphylococcus aureus were labeled with fluorescein‐isothiocytanate (FITC), stained by ethidium bromide (EB), and measured by flow cytometry (FCM). Bacteria were identified by their FITC‐fluorescence and discriminated from the leukocyte cell nuclei by the much higher EB fluorescence and lower coefficient of variation of the latter. Following phagocytosis, both the bacterial FITC‐and EB‐fluorescence decayed. The mean FITC‐fluorescence was reduced about 20% after 15 min and 30–50% after 60 min. Zymosan particles were labeled by FITC and incubated with leukocytes for 15 min. Phagocytes were sorted by FCM and the zymosan particles were liberated by sonication. Their forward angle light scatter was reduced by about 50.6 ± 2.1% and the FITC‐fluorescence by 8.7 ± 1.0% (mean ± SD). The reduced FITC‐fluorescence and light scatter suggests degradation of proteins, and the decay of EB‐fluorescence degradation of DNA, but the specificity remains to be established. By this method phagocytes from a patient with systemic lupus erythematosus seemed to have a selective defect in DNA degradation, whereas phagocytes from a patient with acute myelogenous leukemia had a low capacity to degrade bacterial proteins. This technique offers opportunities for automatic measurements of bacteria and zymosan particle degradation by phagocytes.
Three‐color immunofluorescence analysis of mouse B‐lymphocyte subpopulations
Authors: Parks D. R. , Hardy R. R. , Herzenberg L. A.
Published: August 2005

We have modified a fluorescence‐activated cell sorter (FACS) to make three independent immunofluorescence measurements on each cell and used this system to study mouse B‐lymphocyte subpopulations. An argon‐ion laser (emitting at 488 nm) excites fluorescein‐and phycoerythrin‐labeled reagents, and a tunable dye laser charged with rhodamine 6G (emitting at 615 nm) excites an allophycocyanin‐labeled reagent. We report simultaneous measurements of IgM, IgD, and the recently‐defined mouse B lymphocyte antigens BLA‐1 and BLA‐2 on splenic lymphocytes of CBA/J mice and mice of the congenic strain CBA/N (which have an X‐linked immunodeficiency [xid]). These data provide information on relationships among the B‐cell populations in CBA/J “normal” mice and the defective CBA/N that could not be derived from one‐ or two‐ color immunofluorescent measurements. We believe this is the first use of allophycocyanin as an immunofluorescence label.
Differential binding of lectins to lymphopoietic and myelopoietic cells in murine marrow as revealed by flow cytometry
Authors: Reimann J. , Ehman D. , Miller R. G.
Published: August 2005

Murine marrow cells were labeled with a panel of fluorescein‐conjugated lectins and analyzed by flow cytometry. All lectins tested [concanavalin A (Con A), phytohemagglutinin (PHA), wheat germ agglutinin (WGA), pokeweed mitogen (PWM), soybean agglutinin (SBA), peanut agglutinin (PNA), lentillectin (LL)], stained a fraction of marrow cells; the fraction was, over a defined concentration range, linearly related to the logarithm of the lectin concentration. The slope of this titration curve was characteristic of the particular lectin tested but independent of the nominal sugar specificity of the respective lectin. Con A‐, PWM‐, and PNA‐labeled marrow cells showed brightly and dimly stained subsets in relative fluorescence intensity distribution histograms at high or intermediate staining concentrations. These subsets were composed of cells with restricted and characteristic size distributions as determined by forward light scatter. Lectin staining patterns of marrow cells from normal mice were compared with those of athymic nude mice and mice with severe combined immunodeficiency (SCID). Normal and nude mice lacked a subset of small cells, dimly stained by Con A and PWM, but brightly stained by PNA. This subset, uniquely defined by the above lectins, appears to correspond to the lymphopoietic marrow compartment.
Fluorescence energy transfer measurements on cell surfaces: A critical comparison of steady‐state fluorimetric and flow cytometric methods
Authors: Szöllósi János , Trón Lajos , Damjanovich Sándor , Helliwell Stephen H. , Arndt‐Jovin Donna , Jovin Thomas M.
Published: August 2005

The energy transfer efficiency E was measured between fluorescein‐conjugated concanavalin A (Con A) and rhodamine‐conjugated Con A bound to homogeneous tissue culture cells, the HK22 murine lymphoma cell line. Results from a flow cytometric energy transfer method (FCET) and two different steady‐state fluorimeter methods were compared. The data were found to be in close agreement after careful correction of the steady‐state fluorimetric measurements for contributions from dissociating ligand. The biological variability of the individual cells with respect to E was calculated using an error propagation analysis and were found to be less than the variability in the absolute amount of ligand binding per cell. FCET has a number of advantages over the fluorimetric measurements using suspensions of cells: (1) relatively labile receptor‐ligand complexes can be measured; (2) the analysis can be restricted to undamaged cells by gating the data collection on the light‐scattering signals; (3) heterogeneous populations of cells with respect to donor and acceptor topology can be distinguished by the correlation of E with other cellular parameters derived from additional signals or combinations thereof; and (4) the dynamics of donor‐acceptor redistribution on subpopulations can be measured.