bluuebunny/crossref_metadata_2025 huggingface dataset:
Human T lymphocyte differentiation antigens: Effects of blood sample storage on LEU antibody binding
Authors: Hensleigh Paul A. , Waters Virginia Bryan , Herzenberg Leonard A.
Published: August 2005

Current studies of human T lymphocytes and their subsets often use quantitative immunofluorescence analysis with monoclonal antibodies against cell surface antigens. With storage of whole blood or separated mononuclear cells for more than a few hours we have found marked changes in lymphocyte analysis using a fluorescence activated cell sorter (FACS). Experiments were done to determine if these lymphocyte changes were influenced by storage temperature and if lymphocytes could be made more stable by addition of culture media RPMI 1640 to whole blood. Optimal conditions found for blood storage were with with addition of 50% RPMI 1640 and with samples held at room temperature (22° C). With these storage conditions, delay on FACS analysis up to 24 hours did not result in spurious results. When blood samples are collected in places remote from the laboratory or when batch analysis of serially collected samples is desirable, excessive storage times should be avoided.
Direct determination of cell diameter, surface area, and volume with an electronic volume sensing flow cytometer
Authors: Schwartz A. , Sugg H. , Ritter T. W. , Fernandez‐Repollet E.
Published: August 2005

A method is described in which the volume, surface area, and diameter of spherical cells can be quickly and easily determined from linear calibration curves using a flow cytometer which measures electronic volume and is equipped with a log amplifier.
Elastic and inelastic light scattering in flow cytometry
Authors: Kerker Milton
Published: August 2005

A review of the fundamental aspects of elastic light scattering suggests that information about the shape and internal structure may best be obtained from signals measured in the backscattering directions. Size information can be most readily extracted from forward‐scattering signals. Spectral analysis of scattered signals with incident white light is a subject that merits further study.

Signals from cells that have been stained with fluorescent dyes are proportional to dye content in the usual flow‐cytometric configuration except in the case of dense, strongly anisometric structures such as sperm cells.

The recent discovery that Raman signals from molecules adsorbed on small silver particles are strongly enhanced suggests the possibility of utilizing this effect for identification of molecular species inside biological cells.
Morphometric synaptology of a whole neuron profile using a semiautomatic interactive computer system
Authors: Saito Kiichiro , Niki Kazuhisa
Published: August 2005

We propose a new method of dealing with morphometric synaptology that processes all synapses and boutons around the HRP marked neuron on a large composite electron micrograph, rather than a qualitative or a piecemeal quantitative study of a particular synapse and/or bouton that is not positioned on the surface of the neuron. This approach requires the development of both neuroanatomical procedures, by which a specific whole neuronal profile is identified, and valuable specialized tools, which support the collection and analysis of a great volume of morphometric data from composite electron micrographs, in order to reduce the burden of the morphologist.

The present report is also concerned with the total and reliable semi‐automatic interactive computer system for gathering and analyzing morphometric data that has been under development in our laboratory. A morphologist performs the pattern recognition portion by using a large‐sized tablet digitizer and a menu‐sheet command, and the system registers the various morphometric values of many different neurons and performs statistical analysis.

Some examples of morphometric measurements and analysis show the usefulness and efficiency of the proposed system and method.
Hoechst 33342 staining of mouse bone marrow: Effects on colony‐forming cells
Authors: Van Zant Gary , Fry C. Glenn
Published: August 2005

We have systematically studied the effect on hemopoietic colony‐forming cells of staining cellular DNA with the bisbenzimidazole dye, Hoechst 33342. Mouse bone marrow cells could be adequately stained in a 30‐60 min incubation with a 5 μM concentration of stain. Flow‐cytometric analysis of stained cells provided cell distributions with coefficients of variation for the G1 peaks of 6% or less under these conditions. We found considerable heterogeneity among hemopoietic colony‐forming cells with respect to the toxicity of the dye. Toxicity in the proliferatively quiescent stem cell population was not changed when the population became proliferatively active. In the sequence of most sensitive to least sensitive, the five progenitors studied could be arranged as follows: CFU‐M, a megakaryocyte colony‐forming cell; CFU‐E, a relatively differentiated erythroid precursor; BFU‐E, a primitive erythroid precursor; CFU‐GM, a granulocyte‐macrophage precursor; and CFU‐S, the spleen colony‐forming cell or hemopoietic stem cell. A staining procedure involving a 30‐min exposure to 5 μM Hoechst 33342 provided optimal staining and no loss in four of the five progenitor populations; the CFU‐M population was diminished by about 50%. We conclude that Hoechst can be regarded as a vital DNA stain for most bone marrow precursor populations, including the hemopoietic stem cell.
Ligand/receptor internalization: A kinetic, flow cytometric analysis of the internalization of N‐formyl peptides by human neutrophils
Authors: Finney David A. , Sklar Larry A.
Published: August 2005

Fluorescence flow cytometry was used to measure the internalization of the fluorescent ligand N‐formyl‐nle‐leu‐phe‐nle‐tyr‐lys‐fluorescein by human neutrophils. The internalization process was monitored by the accessibility of the receptor‐bound fluorescent ligand to quenching following a change in the pH of the extracellular medium from 7.4 to 3.0. In such a pH change, extracellular ligand or fluorescein are quenched immediately (excitation 488 nm). In contrast, intracellular fluorescein (derived from fluorescein diacetate) or intracellular ligand are quenched with half‐times of ∼20 or ∼40 sec, respectively, at 37°C. The fraction of internalized ligand is calculated by resolving the fast and slow components of the quenching process.

Temporal resolution of the internalization process in this system depends upon two factors. We have previously shown that it is possible to examine essentially continuously the kinetics of ligand binding in the nM concentration range without removing the free ligand (Sklar LA, Finney DA, Cytometry 3:161, 1982). We have now modified a Becton Dickinson FACS IV sample head assembly to permit direct addition of reagents into the cell suspension while on‐line. This enables us to change the suspension pH and evaluate internalization with a time resolution of a few seconds. We observe that internalized ligand can be detected within 1 min and that the rate is proportional to the number of receptors occupied. The rate is essentially linear over the first few minutes and ∼60% of the receptor‐bound ligand is internalized after 3 min.
Flow‐cytometric monitoring of intracellular flavins simultaneously with NAD(P)H levels
Authors: Thorell Bo
Published: August 2005

A cytofluorimeter is described, using the combination of Argon UV (351–363 nm) and Argon Blue (488 nm) lasers. The dual excitation makes it possible to monitor simultaneously the redox state of flavins and NAD(P)H as indicative of cell metabolic state. Light scatter, absorption, and staining with exogenous fluorescent dyes can add additional information. Thus, a five‐parameter flow analysis becomes possible. The present paper describes flavin and NAD(P)H measurements on isolated rat liver cells and mouse bone marrow.
Somatic cell genetics and flow cytometry
Authors: Kamarck Michael E. , Barbosa James A. , Kühn Lukas , Peters Pamela G. Messer , Shulman Lester , Ruddle Frank H.
Published: August 2005

Human genes coding cell surface molecules can be introduced into mouse host cells using a variety of somatic cell genetic techniques. Because these human gene products can be detected using indirect immunofluorescene on viable cells, the genes themsleves can be monitored and manipulated using flow cytometry and sorting. In this paper, we review ways that we have used cell sorting to develop a somatic cell genetic analysis of the human cell surface.
Centromeric index measurement by slit‐scan flow cytometry
Authors: Lucas J. N. , Gray J. W. , Peters D. C. , Van Dilla M. A.
Published: August 2005

We report here the application of slit‐scan flow cytometry (SSFCM) in the classification of muntjac, Chinese hamster, and human chromosomes according to centromeric index (CI) and total flurescence. Chromosomes were isolated from mitotic cells, stained with propidium iodide and processed through the SSFCM where flurescene profiles were measured. The centromere for each profile was taken us the point of maximum difference between the measured profile and a standard profile having no centrmeric dip. The areas under the profile on eithr side of the centromere were then calculated and the CI was calculated as the ratio of the larger area to the total area under the profile. Relative DNA contents for each chromosome were taken to be proportioned to the total fluorescence. Mean CI's for muntjac chromosomes 1, 2, and × + 3 were 0.52, 0.88, and 0.73, respectively; CI's for Chinese hamster M3‐1 chromosomes 1, 2, 5, 8, and M2 were 0.53, 0.55, 0.77, and 0.86, respectively; and average CI's for chromosome groups 4 + t (X;5), 6 + 7 + Y, 9 + M1, and 10 + 11 were 0.56, 0.82, 0.82, 0.58, and 0.60, respectively. These results were, on average, within 4.4% of CI measurements made by image cytometry. CI's measured for human chromosomes 9 through 12, were, on average, within 2.0% of those made by image cytometry.
Immunofluorescence analysis of bacillus spores and vegetative cells by flow cytometry
Authors: Phillips A. P. , Martin K. L.
Published: August 2005

A commercially available flow cytometer (Cytofluorograf) was used for the immunofluorescence (IF) analysis of spores of Bacillus anthracis, Bacillus cereus, and Bacillus subtilis, using fluroescein‐labelled antispore conjugates. The cytometer was modified to allow analysis of known numbers of bacteria. In attempting to identify the region of the cytometer fluroescence histogram associated with the presence of stained spores, evidence was produced for signal components due to antibody bound to extracellular antigens. Under some reaction conditions these components were large enough partially or completely to obscure the fluroescence distribution imputed to the spores. The results support the hypothesis that the fluroescence histogram for a bacterial suspension can be modified by subtracting the histogram of the cell‐free centrifugation supernatant to provide a fluorescence distribution more representative of the bacteria themselves. Spore and vegetative forms of B. anthracis could be differentiated in the flow IF assay by comparing the peak and area (integral) values of the photomultiplier output. The 90° scatter histograms of the stained spores and their cell‐free supernatants were so alike in shape that it was not possible to ascrible a unique peak to the spores themselves. Overall, these results confirm the considerable potential of flow cytometry for the rapid and quantitative IF assay of bacterial populations.
Cytofluorometric analysis for estrogen receptors using fluorescent estrogen probes
Authors: Kute Timothy E. , Linville Constance , Barrows George
Published: August 2005

Estrogen receptor (ER) analysis of breast cancer tissue has been shown to be very useful in predicting which patients will respond to hormone therapy and have a better prognosis. The ER assay is, however, tedious and time consuming. Measurement of ER by flow cytometry would be rapid and based on either an average fluorescence‐E2 probe intensity per cell or the percentage of the ER+ cells per cell suspension.

Analysis of E2 modified structures for relative binding affinity to the ER determined by competition studies and for fluorescence uptake into cell suspensions determined by flow cytometry was performed. Lack of high affinity to the ER and purity of the compound were major problems for the fluorescein‐labeled estrogen in fluorescein‐E2 compounds demonstrated by HPLC very little estradiol derivative in the parent compounds compared to total components present. A second type of fluoresceinated estrogen which has a peptide bond between the steroid and the chromophore was also tested. It was less contaminated but was unable to get into the cell and showed no binding activity to the ER. A pure plant fluorescent estrogen, coumestrol, has Ka of 6 × 108 M−1 for the ER and is a single component as determined by HPLC.

Specific fluroescent uptake of coumestrol was performed on ER+ and ER− viable cell suspensions. When these coumestrol‐cell suspensions were excited at 350–360 nm and the blue emission was measured using flow cytometry, the result was a fluroescence uptake that was not highly displaceable by exess nonfluorescence E2 probes. Further studies on the modification of several parameters of the incubation step to enhance specific fluorescent uptake were also unsuccessful. Coumestrol was, however, a good competitor for both cellular and nuclear uptake of 3H‐E2.
Preparation and stability of sixteen murine tissues and organs for flow cytometric cell cycle analysis
Authors: Burns E. Robert , Bagwell C. Bruce , Hinson William G. , Pipkin James L. , Hudson Jerry L.
Published: August 2005

Three different technical protocols were used to prepare samples for flow cytometric (FCM) analysis. Each protocol developed worked best for only certain organs. Protocol I involved mincing small pieces of fresh tissue in the propidium iodide (PI) staining solution and filtering through packed glass wool. The organs that were prepared by protocol I were: submandibular gland, urinary bladder, liver, thymus, bone marrow, spleen, lung, kidney and testis. Protocol II involved exposure of the organ to 0.5% acetic acid for 48 h prior to mincing in the PI. The organs that were prepared by protocol II were: uterus, rectum, colon, ileum, and heart. Protocol III utilized an exposure to 0.5% acetic acid, pepsinization, and then staining with PI. The tissues that were prepared by protocol III were the epithelium of the anterior surface of the cornea and the epithelium of the surface of the tongue. A total of 16 different organs and tissues were successfully prepared. For each organ, averaged DNA histograms were analyzed by nonparametric and parametric programs and the results (phase fractions) are presented in tabular form. Several of the organs used came from animals exposed to 1.0 mg/kg vincristine (VC) for 5–6 h to test the capability of the different protocols to detect the enlargement of the G2 + M compartment by the accumulation of VC‐arrested mitotic figures. The stability of the many different sample preparations was tested by comparing averaged DNA histograms obtained on the day of sample preparation to averaged DNA histograms of the same set of samples after storage at 4°C, with or without fixation in 10% phosphate‐buffered formalin, for days to weeks. After staining with propidium iodide, fixation of the sample with a final concentration of 2–3% phosphate‐buffered formalin, was the procedure adopted to assure sample stability. The demonstration of sample stability permits sample preparation to occur at one site followed by transport of the samples to the FCM laboratory at another geographical location. The major findings of this work were (a) technical protocols were developed which resulted in acceptable nuclear suspensions for FCM from 16 different murine organs or tissues, (b) the stability of these samples can be assured by fixing the PI stained nuclear suspension with formalin, and (c) each different protocol was capable of detecting and preserving at least some of the mitotic figures arrested and collected by vincristine.
The use of trout erythrocytes and human lymphocytes for standardization in flow cytometry
Authors: Jakobsen A.
Published: August 2005

A method of standardization of flow cytometric ploidy measurements using trout erythrocytes and human lymphocytes is described. The sources of errors of the ratio between the modal channel number of erythrocytes and lymphocytes were investigated. The sample standard deviation was 0.3%, and the variation between persons of the same sex was approximately 0.5%. A distinct difference (1.82%) between the two sexes indicated that small deviations of DNA content can be detected.

The standardization method was applied to the analysis of biopsy specimens from bladder tumors, normal bladder mucosa, and lymphocytes from the same patients. In diploid populations the standard deviation of the DNA indices was 1.5–2% for normal bladder mucosa but approximately 2.5% for tumors. These values indicate that deviations amounting to 4–5% from the diploidy are detectable by a single analysis of one sample.
Maturation rates and transition probabilities of cycling cells
Authors: Sahar Elhanan , Wage Michael L. , Latt Samuel A.
Published: August 2005

The kinetics of cell cycling are analyzed in terms of equations incorporating a DNA synthesis rate continuously varying along S phase and a portion of G1 phase through which the cells advance in a probabilistic fashion. These equations are solved analytically for several steady state as well as nonsteady state populations of cells. Experiments are described illustrating how these results can be applied to obtain both the rate of DNA synthesis at each point along S‐phase and the probability of transition through the nondeterministic portion of G1.
Increased uptake of vital dye hoechst 33342 during S phase in synchronized HeLa S3 cells
Authors: Frankfurt Oskar S.
Published: August 2005

Uptake of the vital dye Hoechst 33342 (HO342) was studied in HeLa S3 cells synchronized with hydroxyurea or thymidine. Cell monolayers were incubated with 0.05–0.5 μg/ml HO342 for 1–10 min at 37°C, and the fluorescence intensity of DNA‐bound intracellular dye was then measured in a flow cytometer. Modal fluorescence intensity normalized to DNA content was used as a measure of the dye uptake. No efflux of the dye from vitally stained cells was observed during 2 hr at 37°C in a monolayer or at 20°C in a suspension. HO342 uptake increased within in 1 hr after the renewal of progression (early S phase), achieved a maximum level in 2 hr (mid‐S phase) and returned to the inital level in 4 hr (late S‐G2 phase). The pattern of increased uptake of HO342 was similar in cells synchronized with either hydroxyurea or with thymidine. To estimate the relationship between fluorescence and dye uptake, the amount of dye released by Triton X‐100 after vital staining was measured. The method for this measurement was based on the simultaneous addition of chicken erythrocytes (“indicator cells”) with Triton X‐100. Fluorescence intensity of chicken erythrocytes was significantly higher in the mixture containing asynchronous or late S‐G2 cells. Thus the fluorescence after vital staining with HO342 reflected HO342 uptake and, possibly, fluctuations of membrane permeability. The period of maximal HO342 uptake in synchronized cells was also characterized by a a high sensitivity of cytotoxic effects of adriamycin. Uptake of HO342 at nonsaturating concentrations may be used as a marker of cell sensitivity and resistance to a number of cytotoxic compounds.
A rapid, vital staining procedure for flow cytometric analysis of human reticulocytes
Authors: Sage Burton H. , O'Connell James P. , Mercolino Thomas J.
Published: August 2005

A fluorescent staining procedure based upon thioflavin T is described. This dye permits rapid, vital, nonprecipitative staining of human reticulocytes. Flow cytometric analysis of thioflavin T stained whole blood specimens results in reticulocyte counts that correlate well with new methylene blue reticulocyte counts and give coefficients of variation approaching the theoretical limit. The shape of the fluorescence histogram is qualitatively similar to the reticulocyte age profile. Paired new methylene blue and thioflavin T reticulocyte counts are presented with illustrative cases of reticulocyte maturity distributions.
Analysis of malaria parasite‐infected blood by flow cytometry
Authors: Jacobberger James W. , Horan Paul K. , Hare J. Donald
Published: August 2005

The use of flow cytometry in the quantitative analysis of blood from mice infected with Plasmodium vinckei has been studied. Several fluorescent dyes responsive to cell membrance potential were screened and one dye, 3,3′‐dimethyloxacarbocyanine (DiOC1(3)), was chosen for further study. Mature red blood cells (mRBC), immature RBC (imRBC), and parasitized RBC (pRBC) could be recognized and counted in the flow cytometer. When infected blood was separated on a Percoll gradient and fractions analyzed by flow cytometry using DiOC1(3), distinct populations of pRBC were recognized, the frequency of which varied with density. These subpopulations could not be correlated with distinct morphologic stages but varied with the size or age of the growing parasite. Methods combining the use of DiOC1(3) with a DNA specific‐dye, Hoechst 33342, are discussed as an approach to more complete analysis of the blood of malaria‐infected animals.
The mutual effect of hydrogen ion concentration and osmotic pressure on the shape of the human erythrocyte as determined by light scattering and by electronic cell volume measurement
Authors: Porath‐Furedi Asher
Published: August 2005

The measurement of the fluctuations of the scattered light were the source of information on the flatness of erythrocytes. Data on cell volume, together with the measure of scattered light fluctuations, defined the cell shape. The measurements were repeated in a wide range of osmotic pressures and pH values in order to affect both the hemoglobin and the cytoskeleton. Major volume changes were detected at low osmotic pressures and pH. The erythrocyte volume was determined by electronic volume measurement. The cell flatness is maximal at physiological conditions.
Multidimensional slit‐scan prescreening system: Preliminary results of a single blind clinical study,
Authors: Wheeless L. L. , Patten S. F. , Berkan T. K. , Brooks C. L. , Gorman K. M. , Lesh S. R. , Lopez P. A. , Wood J. C. S.
Published: August 2005

A multidimensional slit‐scan flow system was developed to serve as an automated prescreening instrument for gynecological cytology. A 2‐year single blind clinical study was carried out to evaluate system performance. Cellular material was collected by scraping the uterine cervix and stained in suspension with acridine orange. Seven hundred and forty specimens (701 patients) including 156 abnormal specimens representing a broad spectrum of abnormality were analyzed. Approximately 50,000 cells were analyzed for each specimen. The system false‐positive rate was 17.6% while the false‐negative rate was 2.8%. All misclassified abnormals were specimens with cellular changes consistent with a slight dysplasia of nonkeratinizing type. The instrument in its present configuration appeared sensitive to the entire spectrum of abnormality existing in the female genital tract and it classified as abnormal any specimen containing on the order of 0.1% (or greater) abnormal cells.
Erythrocyte deformability and size measured in a multiparameter system that includes impedance sizing
Authors: Bator Jenny M. , Groves Michael R. , Price Brandon J. , Eckstein Eugene C.
Published: August 2005

A Coulter Counter was modified to allow the photographing of erythrocytes as they exit the sizing orifice. These photographs provide the necessary information to determine the shape of individual cells. This data was combined on a cell‐by‐cell basis with the corresponding impedance signal to determine accurately both the cell size and deformed state of each cell. The particle shape is required if the size is to be determined accurately. Normal, diabetic and sickle cell samples were used in this study. The cell size was corrected according to theory that relates the impedance signal with cell shape. Although there was no objective test to determine the efficacy of this calculation in terms of improving the size measured, the reduction in the associated distribution width is taken as indicative of an improvement in the measuring process.

The data presented represent the first attempt to relate cell size with deformed state on a cell‐by‐cell basis and indicates the potential usefulness of multiparameter deformability and size measurements.
Filipin as a flow microfluorometry probe for cellular cholesterol
Authors: Muller Claude P. , Stephany David A. , Winkler David F. , Hoeg Jeffrey M. , Demosky Stephen J. , Wunderlich John R.
Published: August 2005

The polyene antibiotic filipin, which forms specific complexes with 3β‐hydroxysterols, displays spectral properties compatible with its use in flow microfluorometry (FMF). The purpose of this study was to test the suitability of filipin as an FMF probe for unesterified cellular cholesterol. The following experimental conditions appeared optimal for cells with an average unesterified cholesterol content of less than 3 nmol per 106 cells: 2 × 106 fixed cells (1–4% p‐formaldehyde, 30 min, 21°C) stained for 2–4 h with 100 μg/ml filipin and excited at 350. 7/356. 7 nm. Fluorescence emission (Em) was measured above 510 nm. Less suitable conditions involved excitation at 488 nm or using cells which had not been fixed. Fixation preserved the live‐dead cell discrimination provided by forward light scatter measurements, so that dead cells could be excluded from the FMF analysis of cellular cholesterol. Under the above conditions FMF showed that in all cases the fluorescence intensity of filipin‐stained cells was clearly increased above autofluorescence levels of the unstained control cells. The increase in fluorescence signal in different filipin stained cell types correlated (P ⩽ .001) with the cellular content of unesterified cholesterol determined by an independent enzymatic assay. The sensitivity of the FMF assay was in the femtomole (10–15) range. Mixing experiments with cells of different cholesterol levels showed that the technique distinguishes cell populations with distinctive levels of unesterified cholesterol. We therefore concluded that filipin is a useful FMF probe for determining relative levels of unesterified cholesterol in cells.
Flow cytometric analysis of DNA aneuploidy in primary and metastatic human solid tumors,
Authors: Frankfurt O. S. , Slocum H. K. , Rustum Y. M. , Arbuck S. G. , Pavelic Z. P. , Petrelli N. , Huben R. P. , Pontes E. J. , Greco W. R.
Published: August 2005

DNA histograms were measured by flow cytometry for 656 human solid tumors (365 primary and 291 metastatic). The proportion of aneuploid cells in cell suspensions obtained by mechanical disaggregation was significantly higher than those obtained after enzymatic disaggregation (collagenase + DNAse) of the same tumor. A strong correlation was observed between the values of DNA‐indices measured after staining with propidium iodide and with 4′,‐6‐diamidino‐2‐phenylindole (r = 0.97). Aneuploid cells were observed in 430 tumors (66%); 30 of these had two aneuploid stemlines, and two had three aneuploid stemlines. The overall frequency of aneuploidy was 61% among primary and 71% among metastatic tumors. The median value of the DNA index was 1. 67 for 224 primary aneuploid tumors and 1.68 for 206 metastatic aneuploid tumors. For most diseases, the largest proportion of aneuploid primary and metastatic tumors had DNA‐indices in the hypertriploid region. No major differences in frequency and degree of aneuploidy was observed between primary and metastatic tumors. For carcinomas of the bladder and prostate, frequency of aneuploidy was higher among poorly differentiated, than among moderately and welldifferentiated tumors. For carcinomas of the breast and for sarcomas, tumors with DNA indices of > 2.0 were observed mostly in the poorly differentiated group. For patients with carcinomas of the bladder and prostate most tumors at earlier stages of disease were diploid; whereas most tumors at later stages of disease were aneuploid. For patients with carcinomas of the ovary, colon, and kidney, no relationship between stage of disease and aneuploidy was evident.
Development and use of metaphase chromosome flow‐sorting methodology to obtain recombinant phage libraries enriched for parts of the human X chromosome
Authors: Lalande Marc , Kunkel Louis M. , Flint Alan , Latt Samuel A.
Published: August 2005

Metaphase chromosomes isolated from human lymphoblastoid cell lines containing structurally abnormal X chromosomes have been stained with the bisbenzimidazole dye Hoechst 33258 and analyzed on a FACS II flow system equipped with a 5‐W all‐lines argon ion laser. The chromosomal fluorescence has been highly resolved at flow rates of 1,000–3,000 chromosomes per second.

With the goal of obtaining recombinant DNA libraries from parts of the human X chromosome, fluorescence populations enriched for a dicentric X (Xpter‐> Xq24::Xq24‐> Xpter) chromosome and an isochromosome of the long arm of the X [i(Xq)] have been identified. The dicentric X chromosome has been resolved as a discrete peak in the fluorescence flow histogram. In contrast, the fluorescence intensity of the isochromosome is indistinguishable from that of chromosomes 3 and 4. Recombinant DNA libraries from the flow‐sorted chromosomes have been constructed in the lambda phage, Charon 21A, and consist of 1.6 × 105 and 0.7 × 105 plaque‐forming units in the case of the dicentric X and the isochromosome, respectively. Ninety percent of the phage in both recombinant libraries contain inserts which hybridize to highly repetitive human DNA sequences. The recombinant phage library from the flow‐sorted dicentric X chromosome, which could be assigned to a discrete fluorescence peak, has been further characterized and shows at least a tenfold enrichment for X chromosome‐specific DNA sequences as determined by Southern blot hybridization of cloned fragments.
Preparation of chromosome suspensions for flow cytometry
Authors: Engh Ger Van Den , Trask Barb , Cram Scott , Bartholdi Marty
Published: August 2005

Certain variables in the preparation of chromosome suspensions for flow cytometric analysis have been investigated. The optimal conditions have been determined. The results of this series of experiments have been incorporated to yield a preparation protocol that gives chromosome profiles with a low amount of small particle debris and few chromosome clumps. The method reduces variability that results from sample preparation. Chromosomes are optimally isolated in a hypotonic solution buffered to pH 8.0. MgSO4 and dithiothreitol added to the buffer reduce the number of clumps and small fluorescent particles. The presence of MgSO4 also stabilizes the chromosomes and precludes the need for other stabilizing agents such as propidium iodide. When the swelling buffer developed in this investigation is used, unstained chromosomes are stable for at least 1 wk if stored at 4°C. The preparation procedure can be used with the DNA stains, propidium iodide, Hoechst 33258, and mithramycin. Preliminary experiments show that this procedure can also be used for bivariate analysis of human and Chinese hamster chromosomes. The importance of this improvement for studies on chromosome damage caused by irradiation or mutagens is discussed.
Flow cytometric analysis of small DNA content differences in heterogeneous cell populations: Human amniotic fluid cells
Authors: Koch H. , Bettecken T. , Kubbies M. , Salk D. , Smith J. W. , Rabinovitch P. S.
Published: August 2005

Accurate flow cytometric determination of nuclear DNA fluorescence has been attempted with human amniocentesis materials employing a detergent nuclear isolation protocol and 4,6‐diamidino‐2‐phenylindole (DAPI) staining. Assay of native fluids yielded inadequate fluorescence histograms, whereas analysis of cultured amniocytes yielded unimodal histograms in 80 of 83 samples. Three fluids displayed unexpected bimodality, which may have resulted from the interaction between detergent and chromatin in rare cell type. Using a standardized protocol for preparation, staining, and assay, including brief periods of cocultivation of a human triploid reference standard, the percent experimental error in the flow cytometric DNA content estimate was as low as 0.2% in repeated assays of a single sample, and 0.4% in assays of a given genotype at different occasions. In two series of blindly assayed specimens, the percent standard error of the DNA content estimate ranged from 0.6 to 1.0%.
Correlation between protein synthesis rate and nuclear morphology of human erythroblasts as determined by quantitative autoradiography and high‐resolution image analysis
Authors: Dörmer P. , Abmayr W. , Giaretti W.
Published: August 2005

Human erythropoiesis is an appropriate model for analyzing correlations between functional activity of cells during maturation and nuclear morphology as determined by high‐resolution image analysis. In a previous study a relationship had been found between nuclear condensation and the proliferative activity of the cells. This present study analyzed the correlation between nuclear morphology and the protein synthesis rate. The latter was assayed by measuring the 14C‐I‐leucine incorporation rate utilizing the technique of quantitative 14C‐autoradiography. The labeled cells were first classified according to conventional cytological criteria into four groups of increasing maturity. Following grain counting the nuclei were Feulgen‐stained, and after removel of the silver grains, the local nuclear optical densities were evaluated by scanning.

There was a nonlinear relationship between the protein synthesis rate and features representing the degree of chromatin condensation. This nonlinearity was explained by the mediator function of RNA, predominantly mRNA. The amount of protein produced at a given time depends on the transcriptional activity of the chromatin, the frequency of mitotic divisions partitioning the mRNA, and the half‐life of the mRNA. It was concluded that the chromatin texture of erythroblasts reflects three different metabolic activities: the rate of DNA strand duplication, the transcriptional activity for structural proteins enabling a cell to grow and cycle, and for functional proteins, particularly hemoglobin. Since these three activities appear to be synchronized, it is understandable that functional as well as textural features can be used to perform a supervised classification by means of multivariate analysis. Application of the five most significant features allowed a consistent classification of the erythroblasts into the various cytological compartments in 76.7% of all cases, while the classification error of the observer amounted to 13.7%.
Rare‐event analysis methods for detection of fetal red blood cells in maternal blood
Authors: Cupp James E. , Leary James F. , Cernichiari Elsa , Wood James C. S. , Doherty Richard A.
Published: August 2005

Methods for analyzing very small subpopulations of cells (“rare‐event analysis methods”) are of interest in many areas of biology. In this paper we demonstrate the ability to detect one Rh‐positive erythrocyte per 100,000 Rh‐negative erythrocytes. This is the level needed to detect human fetal red blood cells in the maternal circulation in the case of fetal‐maternal Rh‐incompatibility.

Erythrocytes stained with fluorescent immunobeads are more than 100 times as bright as the same cells stained by indirect immunofluorescence (or almost 40 times as bright as cells stained by the biotin‐avidin technique). More importantly there is greater than a sevenfold increase in signal‐to‐noise (S/N) ratio, which proves critical in detecting very low frequency („rare event”︁) cell subpopulations. Although equal mixtures of positive and negative cells can easily be detected, positives cannot be easily distinguished from negatives when they are present in relative frequencies of 1:1000 or less as measured by either indirect immunofluorescence or biotin‐fluorescent avidin labeling of D antigens. However, use of fluorescent immunobeads and high‐speed flow cytometry allows detection of subpopulations of the same cells at relative frequencies of less than 1:100,000. Based on calibrations using free‐fluorescent immunobeads, the average Rh‐positive cell (when present in a relative frequency of 1:100,000) labeled with 36 ± 19 fluorescent immunobeads. Rh‐positive cells of relative frequencies 1: 10,000 and 1: 100,000 can be detected above background (Rh‐negative red blood cells treated with the same amount of fluorescent immunobeads). Using defined mixtures of Rh‐positive and negative cells, the expected numbers of positive cells were observed over a range of 1:100 to 1:100,000 relative Rh‐positive cell frequencies. These methods should prove of general use in applications where original frequency information about rare cell subpopulations is important and would be lost if enrichment procedures were to be used.
The measurement of specific cell: Cell interactions by dual‐parameter flow cytometry
Authors: Segal David M. , Stephany David A.
Published: August 2005

The Fc receptor‐mediated aggregation of antibody‐coated spleen cells from the P388D1 mouse macrophage line was followed using a novel flow cytometric technique. P388D1 and spleen cells were directly labeled with green‐emitting (fluorescein isothiocyanate) and red‐emitting (substituted rhodamine isothiocyanate) flurophores, respectively. They were mixed, incubated in suspension at 4°C, and analyzed for aggregation with a dual laser flow cytometer. Unconjugated cells appeared as particles which were either red or green, while conjugates were detected as particles which were both red and green. Using this assay procedure, 5 × 104 cells were analyzed in 2‐3 min for the percentages of conjugates, free spleen cells, and free P388D1 cells. Intercellular aggregation required both antibody on the spleen cells and free Fc receptors on the P388D1 cells; nonspecific aggregates accounted for 1% or less of the total particles analyzed. Measurements of the fluorescence distributions within conjugates indicated that the majority of conjugates contained a single P388D1 cell bound to 1–3 spleen cells, and that only heterophilic aggregation occurred. The flow cytometric technique described here should be applicable for the measurement of the initial events of intercellular aggregation in other systems as well.
Cell sorting and microchemistry of cultured human fibroblasts: Applications in genetics and aging research
Authors: Jongkind Johan F. , Verkerk Anton
Published: August 2005

Cell sorting is a way to isolate viable subpopulations of cells present in a mixture. The drawback of the isolation method is the shortage of material for subsequent biochemical determinations. We have employed a combination of (ultra‐) microchemistry and cell sorting to overcome this problem. The methods enable determinations of protein and several enzyme activities on triton extracts of 5,000–10,000 sorted cells. In addition, using ultramicromethods we could determine enzyme activity in single sorted cells. This combination of methods is used for clinical genetic studies on heterozygote detection in Fabry's disease, an X‐linked genetic disease. More‐over, microchemistry is used to study enzyme activities in sorted autofluorescent “aged” fibroblasts.
Anion transport heterogeneity detected by flow cytometric measurement of NBD‐taurine efflux kinetics
Authors: Muirhead K. A. , Steinfeld R. C. , Severski M. C. , Knauf P. A.
Published: August 2005

NBD‐taurine [N‐(7‐nitrobenzofuran‐4‐yl) taurine], a fluorescent substrate for the human erythrocyte anion exchange system, has been used to test the feasibility of making flow cytometric measurements of anion transport in K562 erythroleukemic cells. Cells were preloaded by incubation with 20 μM‐2mM NBD‐taurine, then diluted 10–30‐fold, and efflux was monitored by measuring fluorescence intensity (FL) as a function of time using excitation at 488 nm. The observed rate of decrease in fluorescence was sensitive to temperature and also to phloretin, a compound known to inhibit anion transport and other carrier‐mediated transport processes. The coefficient of variation (CV) of the fluorescence distribution increased markedly over the efflux period, suggesting heterogeneity of the K562 population with respect to the rate constant for NBD‐taurine efflux. This heterogeneity was also reflected in the upward curvature of a first order plot of log (FLt–FL∞) versus time. Half‐times calculated from initial linear portions of the first‐order plots were found to decrease as the loading concentration of NBD‐taurine was decreased, as predicted for a saturable transport system.

NBD‐taurine is not an ideal anion transport substrate for flow cytometric studies. It appears to bind to high‐affinity sites within the cells with consequent fluorescence quenching, complicating interpretation of kinetic curves at low concentrations. However, with the appropriate choice of substrate, flow cytometric estimates of kinetic parameters offer a number of advantages over bulk methods: (1) decreased sample size requirements, particularly at low substrate concentrations, (2) sensitive detection of transport heterogeneity as time‐dependent changes in CV and (3) the possibility of sorting subpopulations with different transport parameters for correlation with state of differentiation or cellular physiology.
Reduced insulin endocytosis in serum‐transformed fibroblasts demonstrated by flow cytometry
Authors: Murphy Robert F. , Powers Scott , Cantor Charles R. , Pollack Robert
Published: August 2005

Neoplastic transformation often results in the loss of growth control and concomitant changes in cell surface properties. The changes in endocytosis of a variety of probes after serum or anchorage transformation were measured for mouse fibroblasts by flow cytofluorometry. No major differences in dextran (fluid phase) or histone (nonspecific‐adsorptive) endocytosis were observed among four cell lines with different growth properties. However, decreased receptor‐mediated internalization of α‐macroglobulin was observed for cell lines transformed to either serum or anchorage independence. Furthermore, increased wheat germ agglutinin and decreased insulin endocytosis were observed, but only in serum transformants. The changes specific to serum transformants were not accounted for by changes in binding of wheat germ agglutinin or insulin. The possible implications of these observations regarding serum transformation and the insulin requirement for growth in serum‐free medium are discussed.
Incorporation of 35S‐sulfate and 3H‐glucosamine into heparan and chondroitin sulfates during the cell cycle of B16‐F10 cells
Authors: Blair Owen C. , Sartorelli Alan C.
Published: August 2005

Changes in glycosaminoglycan composition occurring during the cell cycle were determined in B16‐F10 cells sorted flow cytometrically with respect to DNA content. Incorporation of 35S‐sulfate into heparan sulfate and chondroitin sulfate of unsorted and G1, S, and G2 + M sorted cells was determined following chondroitinase ABC or nitrous acid treatment; the incorporation into surface material was measured as the difference between the radioactivity of control and trypsin‐treated cells. Incorporation of 35S‐sulfate and 3H‐glucosamine into cetyl pyridinium chloride (CPC)‐precipitable material was characterized before and after chondroitinase or nitrous acid treatment by Sephadex G50 chromatography. Long‐term (48 h) and short‐term (1 h) labeling studies demonstrate that (a) the amount of total cellular chondroitin sulfate is greater than that of heparan sulfate, with larger amounts of unsulfated heparan than chondroitin being present; (b) the rate of turnover of heparan sulfate is greater than that of chondroitin sulfate; (c) greatest short‐term incorporation of 3H‐glucosamine into CPC‐precipitable material occurs during S phase; and (d) the rate of turnover of both heparan sulfate and chondroitin sulfate is decreased in S phase relative to G1 and G2+ M.
An ultra‐pure in vitro phase synchrony method employing centrifugal elutriation and viable flow cytometric cell sorting
Authors: Rice G. C. , Dean P. N. , Gray J. W. , Dewey W. C.
Published: August 2005

We present a method of synchronizing cells in G1‐, S‐, and G2M‐phases employing sequential centrifugal elutriation and viable flow cytometric cell sorting of Hoechst‐33342 stained Chinese hamster ovary cells. G1‐ and S‐phase cells can be separated to greater than 99% homogeneity and G2‐M to 70% purity. Most of the 30% contamination in the G2‐M fraction was due to S‐phase cells, whose reproductive integrity could be eliminated through the use of high specific activity 3H‐TdR. There were minimal toxic effects or perturbations to growth following the selection procedures. The most significant limitation of this technique appears to be the rate of cell sorting, which, with current equipment, is approximately 3,000 cells per second.
The use of projections for dimensionality reduction of flow cytometric data
Authors: Mann R. C. , Popp D. M. , Hand R. E.
Published: August 2005

In order to visualize multiparameter flow cytometric measurements it is desirable to reduce the dimensionality of the data to two while preserving important features of each data pattern. We describe a method that projects two‐parameter data onto a straight line that forms an angle θ with one of the original parameter axis. The angle is selected interactively or by principal component analysis. The procedure is applied on‐line or off‐line to pairs of parameters of the original data set that has been collected in LIST mode.
New fluorochromes, compatible with high wavelength excitation, for flow cytometric analysis of cellular nucleic acids
Authors: Latt Samuel A. , Marino Michael , Lalande Marc
Published: August 2005

The spectroscopic properties of three new dyes, EK4, VL772, and LL585, free and bound to nucleic acids, are presented, with particular emphasis on their potential use in flow cytometry. Two of these dyes, EK4 and LL585, exhibit red fluorescence, while dye VL772 exhibits yellow fluorescence. Dye LL585 exhibits specificity for DNA, relative to RNA, and a marked enhancement of fluorescence efficiency upon binding to DNA, needed for a red fluorescent DNA‐specific stain for flow cytometry. The dye penetrates live cells, although uniformity of nuclear fluorescence, as evidenced by DNA flow histograms, is better if the cells are first permeabilized with Triton X‐100. Dye VL772 exhibits yellow fluorescence and little DNA‐RNA discrimination, but may prove useful in conjunction with dye LL585 when simultaneous assay of cellular RNA and DNA is desired. Dye EK4 shares properties of the other two dyes but fluoresces with much less efficiency. Dyes LL585 and VL772, used singly, as a pair, or in combination with blue‐fluorescing DNA specific dyes, such as bisbenzimidazole derivatives, should permit new, convenient analyses of the content and organization of cellular nucleic acids.