bluuebunny/crossref_metadata_2025 huggingface dataset:
Flow cytometric purification of Alzheimer's disease amyloid plaque core protein using thioflavin T
Authors: Palutke Margarita , Kukuruga Debra , Wolfe David , Roher Alex
Published: August 2005

Amyloid plaque core protein (APCP) of Alzheimer's disease obtained from brain tissue homogenate is difficult to recover in pure form, primarily because of contaminating lipofuscin (LF) granules. Thioflavin T, a fluorescent dye previously used to stain amyloid, was found to bind to APCP but not to lipofuscin. The latter, however, is autofluorescent. Fluorometric studies showed that at 370 nm excitation APCP has a maximal emission at 418 nm, whereas the autofluorescent LP has a maximal emission at 450 nm. This difference in emission permitted the use of a flow cytometer‐sorter (FACS 440) for purification of APCP. APCP particles fluoresced distinctly from LF granules on the log blue fluorescence parameter. The two entities were sorted using forward light scatter versus fluorescence. A collection apparatus was designed and prepared to facilitate the collection of large volumes of sheath fluid and particles and to minimize fragmentation of particles during the collection process. The sorted APCP fraction was 98% pure. This work demonstrates how old dyes can be used to perform new tricks and provide a useful method for separating complex protien.
Immunofluorescent quantification of ribonucleotide reductase M1 subunit and correlation with DNA content by flow cytometry
Authors: Mann Graham J. , Dyne Marlen , Musgrove Elizabeth A.
Published: August 2005

The cytoplasmic enzyme ribonucleotide reductase is essential for DNA synthesis, and its activity is strongly correlated with cellular proliferation. This paper describes a flow cytometric technique for the simultaneous measurement of DNA content and the M1 subunit of ribonucleotide reductase. Data are presented for cycling cultured human leukemic lymphoblasts in which M1 is constitutively expressed, and peripheral blood lymphocytes in which it is only detectable with certainty after mitogen stimulation.

The choice of fixation procedure strongly influenced the amount of M1 subunit detected. Paraformaldehyde (PF) at concentrations of 2% (w/v in PBS) or greater provided optimal results. Fixation at 37°C was significantly more effective in preserving M1 than fixation at room temperature or 4°C. These variables are shown to have affected cytoplasmic retention during postfixation processing. Their relevance to the flow cytometric measurement of other intracellular components by this procedure are discussed.
Spectral analysis of flow cytometric data: Design of a special‐purpose low‐pass digital filter
Authors: Sloot Peter M. A. , Tensen Pieter , Figdor Carl G.
Published: August 2005

Spectral decomposition of flow cytometric datafiles of arbitrary dimension reveal information of both the signal and the noise components that constitute the histograms. This spectral information is used to construct a low‐pass digital filter, which removes the high‐frequency noise from the actual data. It is shown that this procedure guarantees nontrivial smoothing of the flow cytometric data in accordance with the local experimental situation. As a consequence optimal reconstruction of the signal is possible, which facilitates unambigous interpretation of the data files and mathematical estimation of the statistical parameters.
Flow cytometric analyses of lectin binding to rat alveolar macrophages
Authors: Saunders George C. , Steinkamp John A. , Lehnert Bruce E.
Published: August 2005

The binding characteristics of ten FITC‐labeled plant lectins (Con‐A, MPA, BPA, PNA, WGA, SBA, UEA‐I, DBA, GS‐I, GS‐II) to lavaged rat alveolar macrophages were assessed by flow cytometry. The alveolar macrophages (AM) were incubated with varying concentrations of each lectin in a pinocytosis‐inhibiting buffer. In addition to measuring lectin‐associated green fluorescence, the electronic cell volumes and axial light loss characteristics of the AM were also measured flow cytometrically. These latter parameters were found to be good indicators of cell agglutination caused by some of the lectins, and, in conjunction with green fluorescence measurements, usefully serve to determine optimal or nonagglutinating lectin concentrations for flow cytometric studies. With the exception of UEA‐I, all of the lectins examined bound to AM, although a wide range of binding was observed among the lectins. At subagglutinating concentrations, Con A, MPA, BPA, PNA, WGA, SBA, and GS‐I bound to the AM with unimodal patterns. Histograms of lectin‐associated fluorescence intensity obtained with DBA clearly presented a pattern consistent with a more complex, bimodal distribution of labeled AM, suggesting the presence of at least two subset populations. The low‐intensity distribution of AM represented ≃ 70% of the cells, while the more strongly labeled subset represented ≃ 15% of the parent AM population. The remaining balance of the AM was identified as another subpopulation by the failure to detectably bind to the DBA. While GS‐II bound to all of the AM, this lectin labeled about 5% of the cells much more intensely than the bulk of the population. Thus, two subset populations of AM could be resolved according to their differing avidities for the GS‐II lectin.
Improved program for the analysis of DNA histograms
Authors: Ormerod M. G. , Payne A. W. R. , Watson J. V.
Published: August 2005

We describe a computer program which will rapidly analyze a set of DNA histograms without further operator intervention after the initial input. The algorithm used was modified from that of Watson et al. (Cytometry 8:1–8, 1987). The program, coded in Pascal, has been implemented on two different systems. It has been tested by using simulated data and experimental data from cells labelled with bromodeoxyuridine.

The program has been in use in this and other laboratories for over two years and has proved to be robust and fast and to give a reasonable approximation to the experimental data. It is particularly useful when dealing with badly perturbed DNA histograms.
Four‐Parameter white blood cell differential counting based on light scattering measurements
Authors: Terstappen L. W. M. M. , DeGrooth B. G. , Visscher K. , VanKouterik F. A. , Greve J.
Published: August 2005

Measurement of the depolarized orthogonal light scattering in flow cytometry enables one to discriminate human eosinephilic granulocytes from neutrophilic granulocytes. We use this method to perform a four‐parameter differential white blood cell analysis.

A simple flow cytometer was built equipped with a 5‐mW helium neon laser that measures simultaneously four light scattering parameters. Lymphocytes, monocytes, and granulocytes were identified by simultaneously measuring the light scattering intensity at angles between 1.0° and 2.6° and angles between 3.0° and 11.0°. Eosinophilic granulocytes were distinguished from neutrophilic granulocytes by simultaneous measurement of the orthogonal and depolarized orthogonal light scattering.

Comparison of a white blood cell differentiation of 45 donors obtained by the Technicon H‐6000 and our instrument revealed good correlations. The correlation coefficients (r2) found were: 0.99 for lymphocytes, 0.76 for monocytes, 0.99 for neutrophilic granulocytes, and 0.98 for eosinophilic granulocytes. The results demonstrate that reliable white blood cell differentiation of the four most clinically relevant leukocytes can be obtained by measurement of light scattering properties of unstained leukocytes.
Simultaneous cytometric analysis for the expression of cytoplasmic and surface antigens in activated T cells
Authors: Hayden Georgina E. , Walker Karen Z. , Wotherspoon John S. , Raison Robert L. , Miller Jacques F. A. P.
Published: August 2005

A method of two‐colour immunofluorescence staining has been developed to allow the simultaneous analysis of both surface and cytoplasmic antigens. This involves the use of direct fluorochrome antibody conjugates for cell‐surface antigen staining, followed by cell permeabilization and the staining of cytoplasmic antigens with biotinylated antibodies and streptavidin‐fluorochrome conjugates.

Fluorochrome‐antibody conjugates bound to cell‐surface epitopes were found not to be affected by the subsequent permeabilisation and cytoplasmic staining. This method was used to examine the surface phenotype of T cells expressing a cytoplasmic antigen, STA. STA is a unique determinant detected in activated human T cells by the monoclonal antibody K‐1‐21, which also recognizes a cross‐reactive conformation‐dependent epitope on human free kappa light chains. Cytometric analysis showed that STA is found in both Leu 2a+ cytotoxic/suppressor T cells and Leu 3a+ helper/inducer T cells but is not induced in the Leu 15+ population which contains suppressor T cells. STA was also shown to be an activation antigen in murine T cells.
Analysis of simian virus 40 infection of CV‐1 cells by quantitative two‐color fluorescence with flow cytometry
Authors: Lehman John M. , Laffin Judith , Jacobberger James W. , Fogleman David
Published: August 2005

Quantitative two‐color fluorescent analysis of Simian virus (SV40) infection of permissive CV‐1 cells was investigated. Analysis included by quantitation of cellular DNA, the early viral tumor (T) antigen with a monoclonal antibody, and late viral (V) antigens with a polyclonal antibody. T antigen was detected in all phases of the cell cycle at 6 and 12 h, after SV40 infection of growth arrested cells. At later time intervals, the percentage of T‐antigen‐positive cells increased with the induction of the cells into successive rounds of DNA synthesis and an increase in tetraploidpolyploid cells. The amount of T antigen per cell increased as the cells entered the successive stages of the cell cycle (G0/G1 → G2 + M → tetraploid S and G2 + M). The V antigen from adsorbed virus was detected immediately after infection. Synthesis of V antigen began in late S and G2 + M phases of the cell cycle. This quantitative analysis allows a definitive determination of antigen per cell in a population correlated with the cell cycle and may be useful in correlating viral and cellular events with transformation.
Transmission electron microscopy of small numbers of sorted cells
Authors: Sebring R. J. , Johnson N. F. , Spall W. D.
Published: August 2005

A new method that allows the transmission electron microscopic examination of as few as 1 × 104 cells obtained by flow cytometric sorting is described. The approach involves “sandwiching” fixed cells in an agarose case by a microcentrifugation system consisting of small‐diameter cell‐centrifugation tubes and subsequent processing of the cells by conventional techniques. The advantages offered by this method are discussed.
Acousto‐optic laser‐scanning cytometer
Authors: Burger Douglas , Gershman Russell
Published: August 2005

An instrument has been developed that uses a computer‐controlled rapidly scanning laser beam to make cytometric measurements on cells or particles and which can measure low levels of fluorescence when using low‐power lasers (Gershman, Hoffman, and O'Connell, “Methods and Apparatus for Analysis of Particles and Cells.”) The method used is based upon acousto‐optic principles of light diffraction. A vertically polarized 5‐mW He‐Ne laser is directed into an acousto‐optic Bragg cell in which a portion of the incident light under goes a small angular variation or deflection. Suitable optics focus the beam to a 25 μm diameter spot, at the 1/e2 point, in a sample cuvette while translating the angular variation into a linear scan. The cuvette enclosing the sample is slowly moved (approximately 1 μm/ms) via a stepper drive into the scanning beam while the forward angle light scatter sensor is monitored for the presence of valid signal events. When an event occurs, appropriate software optimizes the position of the focused laser beam onto the cell. Subsequently, scanning is stopped to allow for cell interrogation times that last for milliseconds or longer.
Discrimination of myogenic and nonmyogenic cells from embryonic skeletal muscle by 90° light scattering
Authors: Yablonka‐Reuveni Zipora
Published: August 2005

A myogenic cell suspension was isolated from the breast muscles of 10‐day‐old chicken embryos by trypsin digestion. The cell preparation was subjected to Percoll density centrifugation to reduce the number of fibroblastlike cells present. The Percoll‐isolated, predominantly myogenic cell population was then fractionated by flow cytometry using 90° light scattering as the parameter for sorting. Cells exhibiting lower scatter, with a peak of 45 units, produced cultures containing myotubes and gave rise only to myogenic clones. Cells exhibiting higher scatter (120‐200 units) produced nonmyogenic cultures and gave rise to nonmyogenic clones. Cells with intermediate light scatter were also detected. The latter produced both myogenic and nonmyogenic clones. The differences in light scatter presumably reflect higher cytoplasmic complexity of the nonmyogenic cells compared with the myogenic cells. Moreover, the differences in light scattering properties of the different cell types offer a means for the isolation of pure populations of myogenic cells directly from the intact muscle.
Flow cytometric measurements of cytoplasmic calcium changes in human platelets
Authors: Davies Theresa A. , Drotts Daniel , Weil Gary J. , Simons Elizabeth R.
Published: August 2005

Thrombin‐induced stimulation of human platelets is accompanied by a dramatic increase in cytoplasmic calcium concentrations followed by a slow decrease. These changes are very rapid, are maximal by 10‐5 s, and can be detected with probes such as Indo‐1. Suspension studies using spectrofluorometry, which reflect a value which is the average of 3 x 107 cells per ml, indicate a thrombin dosedependent increase in cytoplasmic calcium at doses up to 0.025 units per ml. We show here, using flow cytometry, that at less than halfsaturating thrombin doses only subpopulations of platelets rather than the entire sample are responding. The extent of these responses, however, still depends on thrombin concentration. When the thrombin doses are between half and fully saturating, one subpopulation responds fully (i.e., its extent of increase in cytoplasmic calcium concentration, [Ca++]in is 100% of that seen at saturating thrombin concentrations) while the remaining platelets respond partially or not at all. There is thus evidence of positive cooperativity leading to disproportionate thrombin receptor occupancy on different subpopulations when platelets are subjected to subsaturating doses of thrombin. The existence of responding subpopulations may explain how the reported multiple stimulations of the same suspension of platelets at low thrombin doses occur.
Comparison of fresh and paraffin‐embedded tissue as starting material for DNA flow cytometry and evaluation of intratumor heterogeneity
Authors: Kallioniemi Olli‐Pekka
Published: August 2005

Flow cytometric analysis of DNA ploidy and S‐phase fraction (SPF) from paraffin‐embedded tumors has become an important diagnostic and prognostic tool in clinical pathology and investigative oncology. The present study aimed at elucidating the reliability of the method. About 90% of the 1,400 paraffine‐mbedded tumors analyzed were evaluable for DNA index and about 70% for SPF, although considerable differences between various tumor types were detected. The within‐assay coefficients of variation for determination of tumor DNA index and SPF were 2% and 6%, respectively. Intratumor variation in DNA index was observed in 24% of breast and in 21% of ovarian carcinomas and variation in SPF in 36% and 29% of the respective tumors. Intratumor variation in SPF was greatest in DNA‐diploid tumors, which may indicate that SPF values in these tumors may be less reliable owing to variations in the proportions of tumor and nontumor cells. If the methodological variation and the intratumor variation were taken into account, there was a good correlation between DNA indices (r = 0.980) and between SPF values (r = 0.794) obtained from fresh and paraffin‐embedded tumors. It is concluded that accurate determination of DNA index and SPF from paraffin‐embedded tumors is possible in the majority of cases. Regardless of the type of starting material used for DNA flow cytometry it is advantageous to study several samples from each tumor to account for the intratumor heterogeneity.
Flow cytometric discrimination of mitotic nuclei by right‐angle light scatter
Authors: Zucker Robert M. , Elstein Kenneth H. , Easterling Robert E. , Massaro Edward J.
Published: August 2005

Flow cytometry has been used to demonstrate alterations in protein, RNA, and DNA content of cells as they traverse the cell cycle. Employing fluorescein isothiocyanate (FITC) to stain protein and propidium iodide (PI) to stain nucleic acids, multiple regions within the G1 and G2 phases of the cell cycle, in addition to the M phase, can be distinguished. In this study, cytograms of the 90° light scatter signal vs. PI fluorescence were remarkably similar to those of FITC fluorescence vs. PI fluorescence, suggesting a relationship between 90° light scatter and protein content. M‐phase nuclei can be distinguished from G2‐phase nuclei on cytograms of 90° light scatter vs. PI fluorescence. However, the percentage of mitotic nuclei obtained by this technique is less than that found by light microscopic analysis. Flow cytometric parameters of nuclei prepared by nonionic detergent (NP40) lysis in Dulbecco's PBS, Vindelov's buffer, or Pollack's hypotonic EDTA/Tris buffer were compared. The best resolution of mitotic nuclei was obtained in Pollack's buffer. However, the stainability of the M‐phase nuclei is reduced, and the nuclei are located in the late S/G2 region of the single‐parameter histogram.
Detection of an early cytomegalovirus antigen with two‐color quantitative flow cytometry
Authors: McSharry James , Laffin Judith , Fogleman David , Lehman John M. , Elmendorf Sarah
Published: August 2005

An early cytomegalovirus (CMV) antigen was detected with a monoclonal antibody by two‐color fluorescent flow cytometry. With the aid of a human diploid fibroblast cell strain, FLOW 2000, infected with the AD169 strain of CMV, the viral antigen and the DNA content of infected or uninfected cells were measured. There was no evidence of change in the cell‐cycle distribution of the infected cells. The viral antigen was detected within 30 minutes following virus adsorption at 0.1 and 1.0 plaque‐forming units/cells; and the percentage of positive cells increased with time and viral dosage. All stages of the cell cycle were susceptible to viral infection and the average fluorescence was greater than the background fluorescence. Flow cytometry detected the viral antigen earlier than conventional immunofluorescent microscopy and cell culture for CMV cytopathological effect (CPE). Ten bronchoalveolar lavages assayed by flow cytometry and conventional diagnostic procedures demonstrated that flow cytometry might be useful in early diagnosis for CMV infection.
A correction required for calculation of DNA ratios in flow cytometric analysis of ploidy
Authors: Givan Alice L. , Shenton B. K. , Carr T. W.
Published: August 2005

Flow cytometric measurements of nuclear DNA content often involve the calculation of a DNA index, which compares the DNA fluorescence from two different populations. Such DNA indices have been used to classify aneuploid peaks from tumour tissue into different categories relative to normal diploid cells. This report describes a correction based on the channel for unstained particles that is required if DNA index values are to give a true and reproducible indication of relative DNA content.
Comparison of frequency distributions in flow cytometry
Authors: Cox Christopher , Reeder Jay E. , Robinson Roy D. , Suppes Sarah B. , Wheeless Leon L.
Published: August 2005

A number of methods have previously been considered for the statistical comparison of flow cytometric frequency distributions. For two distributions, the foremost of these is the Kolmogorov‐Smirnov (K‐S) test, which has been criticized as “too sensitive.”

We discuss some alternative methods based on the Poisson distribution. The assumption of Poisson variation within channels allows the use of channel‐by‐channel confidence intervals and chi‐square tests. These are simple and more appropriate for discrete data than the K‐S test. Graphical displays of these and other techniques are presented. We also attempt to set the problem in an appropriate context. We argue that any statistical procedure must rest on a reasonable understanding of the nature of the variability in the system. This understanding takes the form of an appropriate probability model, which may be approximate but must provide a reasonably accurate description of the data. Incomplete understanding of the data can lead to inappropriate analysis. We discuss the assumptions that underlie our techniques and consider extensions to more complex situations.
Identification of cell subpopulations by dual‐color surface immunofluorescence using biotinylated and unlabeled monoclonal antibodies
Authors: Cohen J. H. M. , Aubry J. P. , Banchereau J. , Revillard J. P.
Published: August 2005

A new method of dual‐color immunofluorescence is presented for analysis of surface antigen distribution among heterogeneous cell suspensions. It involves flow cytometric analysis of cells stained with a biotinylated first monoclonal antibody and/or with an unlabeled second monoclonal antibody. After addition of streptavidin‐phycoerythrin and/or fluoresceinated goat antimouse immunoglobulin antibody, single‐cell fluorescence intensties are measured and biparametric graphic representations are obtained, allowing one to determine the percentage of cells stained by each of the monoclonal antibodies or both. The validity of the method was assessed on human peripheral blood mononuclear cells by using three sets of two monoclonal antibodies: CD8 and CD5, CD3 and CD4, CD11 and HLA‐DR. The results showed that dual staining did not induce significant quenching or competition between pairs of antibodies. The procedure is simple and sensitive. It requires only minute amounts of monoclonal antibodies. It is readily applicable to the screening of hybridoma supernatants and to the characterization of new antibodies to cell surface antigens with respect to well‐defined markers.
Chromatin texture changes related to proliferation and maturation in erythrocytes
Authors: Giroud Françloise , Gauvain Catherine , Seigneurin Daneil , Hagen Victoria Von
Published: August 2005

Erythroid cell types were quantitatively assessed on the basis of the analysis of their nuclear images after Feulgen and Heidenhain blue counterstaining. An original nonsupervised classification method, developed in our laboratory, was applied to cell cycle analysis. Results obtained suggest that, in the four proliferative generation cycles, which concur with the successive morphological stages, there is a sequential flow of cells from one morphological stage to the next by cell cycling. We show that the chromatin pattern alone made it possible to set up a metric of the whole differentiation process occurring in the normal human erythroblastic lineage in which proliferating events occur.
Light regulation of the cell cycle in Euglena gracilis bacillaris
Authors: Yee Muh‐Ching , Bartholomew James C.
Published: August 2005

We have studied the light regulation of the cell division cycle in the photosynthetic alga Euglena gracilis bacillaris. Euglena grown under phototrophic conditions are easily synchronized to a 12 h light‐12 h dark regime. By inoculating stationary phase, nondividing cells into fresh media and exposing the diluted cells to either light or darkness, we have determined that initiation of DNA synthesis for the cell division cycle is light dependent. By varying the length of time in light to which synchronized cells are exposed, we have shown that commitment to the cell cycle requires exposure to more than 6 h of light. We propose that this is to allow the accumulation, through photosynthetic electron transport, of an initiating factor that will enable DNA synthesis to begin. Flow cytometry analysis also shows that once cells are committed to the cell cycle, they complete the cycle in the dark, so mitosis is a light‐independent step.
Interval‐coded texture features for artifact rejection in automated cervical cytology
Authors: Tucker James H. , Rodenacker Karsten , Juetting Uta , Nickolls Peter , Watts Keith , Burger Georg
Published: August 2005

In order to improve the separation between abnormal cells and noncellular artifacts in the CERVIFIP automated cervical cytology prescreening system, 22 different object texture features were investigated. The features were all statistical parameters of the pixel density histograms or one‐dimensional filtered values of central and border regions of the object images. The features were calculated for 231 images (100 cells and 131 artifacts) detected as Suspect Cells by the current CERVIFIP and were then tested in hierarchical and linear discriminant classifiers. After selecting the two best features for use in a hierarchical classifier, 83% correct classification was achieved. One of these features was specifically designed to remove poorly focused objects. With maximum likelihood discrimination using all 22 features, an overall correct classification rate of 90% was obtained.
In vitro aging of articular chondrocytes identified by analysis of DNA and tubulin content and relationship to cell size and protein content
Authors: Ronot Xavier , Gaillard‐Froger BÉAtrice , Hainque Bernard , Adolphe Monique
Published: August 2005

In vitro senescence of chondrocytes, characterized by a decline in the proliferation rate during late passages, resulted from a rapid growth rate in early subcultures to a complete loss of division after seven to nine passages. One senescent‐associated phenotypic change was the apparent increase in the density of cytoplasmic cytoskeletal proteins. We examined the relationship between tubulin content and growth (measured by DNA and total protein contents and cell volume), using flow cytometry, in the assessment of cytoskeleton analysis during in vitro aging. In contrast with previous microscopic observations of tubulin organization, flow cytometry revealed a tubulin content that was modulated as a function of protein content and/or cell volume.
DNA flow cytometric analysis of mouse seminiferous epithelium
Authors: Toppari Jorma , Bishop Philippe C. , Parker John W. , Dizerega Gere S.
Published: August 2005

In order to provide a basis for quantitative studies of murine spermatogenesis, we performed a DNA flow cytometric analysis on the mouse seminiferous tubules isolated at defined stages of the epithelial cycle by transillumination‐assisted microdissection. Accurate stage identification was performed by examining spermatids in the adjacent tubule segments by phase‐contrast microscopy. For flow cytometry, suspension of nuclei of spermatogenic cells was obtained by detergent treatment of isolated seminiferous tubules, and fresh samples were stained with propidium iodide.

DNA histograms of the 12 stages of the mouse seminiferous epithelial cycle varied in a stage‐specific manner. DNA histograms of stages I‐VIII of the cycle were characterized by a hypofluorescent haploid peak, the location of which changed with the decreasing DNA dye (propidium iodide)‐binding capacity of elongated spermatids. The absence of the hypohaploid peak and the high ratio of the cells with 4C amount of DNA to the cells with 1C amount of DNA characterized stages IX‐XI of the cycle. Stage XII showed a high 2C peak, owing to a large population of secondary spermatocytes arisen from the first meiotic division. By using fluorescent beads as an internal volume standard cell numbers in defined stages were determined. These data provide a basis for quantitative studies of mouse spermatogenesis.
Flow cytometric detection of ribosomal RNA in suspended cells by fluorescent in situ hybridization
Authors: Bauman J. G. J. , Bentvelzen P.
Published: August 2005

A method using flow cytometry and fluorescent in situ hydridization (ISH) to detect RNA in cells is described. L1210 murine leukemia cells were fixed with 1% formaldehyde in HEPES buffered Hank's balanced salt solution (HH) followed by 70% ethanol. Endogenous RNAses were blocked by diethylpyrocarbonate treatment. Single‐stranded sense and antisense RNA probes, labeled with biotin‐11‐UTP, were transcribed from a 2.1 kb 28S ribosomal RNA (rRNA) gene fragment subcloned into the pGEM2 plasmid. For good results, it was essential that the probes were degraded to 100–150 nucleotides before use. Hybridization was performed at 45°C in 50% formamide, 5 × SSC, 0.5% SDS. Hybrids were detected with streptavidin‐FITC by flow cytometry. Antisense rRNA probe signal was 100 times higher than the background. The hybrids were largely resistant to RNAse and melted at high temperature. The sense probe also gave a signal (5 times background), which was not RNAse resistant and was attributed to the presence of internal inverted repeats in the ribosomal RNA. When sufficient background reduction can be achieved, it is expected that as few as ten mRNA molecules per cell can be detected with the fluorescent in situ hybridization method.
Correction of cellular autofluorescence in flow cytometry by mathematical modeling of cellular fluorescence
Authors: Corsetti James P. , Sotirchos Stratis V. , Cox Christopher , Cowles Judith W. , Leary James F. , Blumburg Neil
Published: August 2005

A method for the correction of background fluorescence in flow cytometry with special relevance to the quantitation of low levels of cellular surface membrane antigens is presented. The method is based on the mathematical modeling of cellular fluorescence distributions of background fluorescence (autofluorescence control or irrelevant antibody control) and total fluorescence (positively stained cells). Algorithms based on two models and utilizing only the routinely available background and total fluorescence histograms are developed and implemented in computer programs. These allow estimation of the fluorescence histogram corresponding exclusively to immunofluorescence staining of the cell surface antigen of interest. Thus, the correction of background fluorescence is effected solely with software processing of routinely available data; no additional hardware or parameter determinations are necessary.

Two models were chosen to be physically plausible and to represent extremes in correlation between background and probe fluorescence. Extremes were chosen to assess the solution dependence on model and to provide bounds to the actual solution when no information on correlation is available. Results are presented for both computer simulations and for an actual assay of the CR1 complement receptor on human erythrocytes to test and illustrate the technique. Alternatively, data can be tested assuming a particular model to explore the relationship, if any, between specific and nonspecific fluorescence.
Comparison between flow cytometry and fluorometry for the kinetic measurement of membrane fluidity parameters
Authors: Collins James M. , Grogan W. McLean
Published: August 2005

Steady‐state fluorescence polarization measurements obtained with a flow cytometer were compared with those obtained with an SLM subnanosecond fluorometer. Measurements were made over time after exposure of HeLa cells to the membrane probe 1,6‐diphenyl‐1,3,5‐hexatriene (DPH), 1‐[4‐(trimethylamino) phenyl]‐6‐phenyl‐1,3,5‐hexatriene (TMA‐DPH), or 12‐(9:anthroyloxy) stearate (12‐AS). After 1 min, anisotropy values of 0.28 (DPH), 0.28 (TMA‐DPH), and 0.21 (12‐AS) were obtained. Thereafter, the anisotropy of DPH‐ and 12‐AS‐labelled cells rapidly decreased (0.18 and 0.12 after 5 min), while that of TMA‐DPH‐labelled cells changed only slightly (0.27 after 30 min), suggesting that DPH and 12‐AS, unlike TMA‐DPH, do not remain anchored in the HeLa plasma membrane, but translocate to more fluid environments inside the cell. These suggestions were confirmed by visual observation with fluorescence microscopy. There was no significant difference between the results obtained with the flow cytometer and those obtained with the fluorometer.
Characterization by flow cytometry of fluorescein‐methotrexate transport in chinese hamster ovary cells
Authors: Assaraf Yehuda G. , Seamer Larry C. , Schimke Robert T.
Published: August 2005

We have studied by flow cytometry the transport of fluorescein‐methotrexate in Chinese hamster ovary cells. Fluorescein‐methotrexate appears to enter cells via a mechanism different from the carrier‐mediated system for methotrexate. This conclusion is supported by the following observations: (1) Fluorescein‐methotrexate is transported equally well into normal and mutant cells defective in the in ward methotrexate uptake. (2) Folic acid and its reduced states, which competitively inhibit methotrexate uptake, do not alter fluorescein‐methotrexate transport. (3) Fluorescein‐methotrexate accumulation exhibits a low temperature coefficient (Q10 = 1.6) compared with the influx of methotrexate (Q10 = 6–8). (4) Initial rates of fluorescein‐methotrexate uptake are concentration dependent but are not saturable. (5) Fluorescein‐methotrexate uptake is very slow and reaches steady state after 8 h, whereas at an equimolar concentration methotrexate reaches saturation after 20 min. (6) Initial influx rates of fluorescein‐methotrexate are not affected by the presence of methotrexate. (7) Sulfhydryl‐reactive mercurials, which block methotrexate transport, do not reduce fluorescein‐methotrexate influx, but rather stimulate it. Thus, based on the nonsaturability of fluorescein‐methotrexate inward transport, its low temperature coefficient, and lack of inhibition with structural analogs, we conclude that fluorescein‐methotrexate is accumulated in hamster cells by a passive diffusion process.
Comparison of fresh, ethanol‐preserved, and paraffin‐embedded samples in DNA flow cytometry
Authors: Alanen Kalle A. , Klemi Pekka J. , Joensuu Heikki , Kujari Harry , Pekkala Erkki
Published: August 2005

Fresh, ethanol‐preserved, and formalinfixed and paraffin‐embedded samples taken from the same part of 15 human tumors, and from one normal spleen and one pancreas were analyzed for nuclear DNA content by flow cytometry. The coefficient of variation (CV) values of the Gl peaks were smaller in the fresh than in the other samples (P < 0.001). The DNA ploidy of the tumors was the same in all types of samples. The DNA indices (DIs) measured from either ethanol‐preserved or formalin‐fixed tissue correlated strongly with those obtained from fresh tissue (P < 0.001), although they tended to be somewhat smaller in the fresh samples. The S‐phase fractions measured from all types of samples were of the same order of magnitude in most cases (P < 0.001). Uninterpretable histograms were most often obtained from fresh samples. Identical DI values and rather constant S‐phase fractions were obtained from ethanol‐preserved samples stored at 4°C for up to 5 months. It is concluded that all three types of samples are suitable for the determination of DNA ploidy, DI, and S‐phase fraction and that 50% ethanol is suitable for long‐term preservation of flow cytometric samples.
Flow cytometry measurements of human chromosome kinetochore labeling
Authors: Fantes Judith A. , Green D. K. , Malloy Patricia , Sumner A. T.
Published: August 2005

A method for the preparation and measurement of immunofluorescent human chromosome centromeres in suspension is described using CREST antibodies, which bind to the centromeric region of chromosomes. Fluorescein isothiocyanate (FITC)‐conjugated antihuman antibodies provide the fluorescent label. Labeled chromosomes are examined on microscope slides and by flow cy tometry. In both cases a dye which binds to DNA is added to provide identification of the chromosome groups. Sera from different CREST patients vary in their ability to bind to ch romosome arms in addition to the centromeric region. Flow cytometry and microfluorimetry measurements have shown that with a given CREST serum the differences in kinetochore fluorescence between chromosomes are only minor. Flow cytometry experiments to relate the number of dicentric chromosomes, induced by in vitro radiation of peripheral blood cells to the slightly increased number of chromosomes with above‐average kinetochore fluorescence did not produce decisive radiation dosimetry results.
Subdivision of S‐phase by analysis of nuclear 5‐bromodeoxyuridine staining patterns
Authors: Van Dierendonck Jan Hein , Keyzer Rob , van De Velde Cornelis J. H. , Cornelisse Cornelis J.
Published: August 2005

After pulse labeling of mammalian cells in vivo or in vitro with 5‐bromodeoxyuridine (BrdUrd), followed by immunostaining with a monoclonal antibody to DNA‐incorporated BrdUrd, various intranuclear staining patterns are observed. These results are obtained in various labeling, fixation, denaturation, and staining conditions. We defined five different patterns in immunoperoxidasestained monolayers of human and rodent cancer cells and compared mean nuclear areas as measured by computerized planimetry. Furthermore, we evaluated frequency distributions of these patterns in partly synchronized cell populations and correlated these results with flow cytometric DNA histograms.

The results indicate that the observed patterns reflect the spatial and temporal organization of DNA synthesis, which seems to be characterized by at least three successive stages of replication. Evaluation of these patterns may have various applications in studies on cell cycle kinetics.
Simultaneous paired analysis by flow cytometry of surface markers, cytoplasmic antigens, or oncogene expression with DNA content
Authors: Dent Georgette A. , Chantal Leglise M. , Pryzwansky Katherine B. , Ross Dennis W.
Published: August 2005

Flow cytometry is a useful tool for measuring DNA content and differentiation as expressed by cell surface markers. We have extended this technology to measure simultaneously either surface, cytoplasmic, or nuclear antigens (particularly oncoproteins) with DNA content. Mononuclear blood cells isolated from normal subjects and HL60 leukemic cells were permeabilized and fixed in suspension utilizing 40 μg/ml lysolecithin and 1% paraformaldehyde. A range of lysolecithin concentrations in 1% paraformaldehyde was studied to optimize permeabilization of the antibodies to the cell interior without destroying cell integrity. The optimal concentration (40 μg lysolecithin/ml) resulted in good cell recovery with a high percentage of cells positive for surface and intracellular antigens. Cells are first stained with fluorescein isothiocyanate conjugated (FITC) anti‐myeloperoxidase (an azurophil granule enzyme), or with an anti‐c‐myc antibody and FITC goat anti‐mouse IgG F(ab')2. Cells are then incubated with RNase and stained for DNA content with propidium iodide. Alternatively, cells were stained for the cell surface markers Leu M3, OKM1, or the transferrin receptor and were then fixed and permeabilized and stained with propidium iodide. Using this method, we correlated cytoplasmic, nuclear, or cell surface antigens with cell cycle kinetics. This technique should be useful for studies of cellular differentiation and proliferation.
Detection of mRNA in flow‐sorted cells
Authors: Dunne J. F. , Thomas J. , Lee S.
Published: August 2005

Cells were sorted onto nitrocellulose filters which were saturated with a lysing cocktail designed to preferentially immobilize cellular mRNA. After washing, these filters were incubated with 32P‐labeled specific DNA probes. We used the phorbol ester/lipopolysaccharide (PMA + LPS) co‐induction of IL‐1 mRNA and CD13 expression in U937 cells to demonstrate the specificity of the technique. In addition we used the abundant expression of c‐fos in U937 to demonstrate linearity. IL‐1 beta mRNA is readily discernable autoradiographically from as few as 5,000 PMA + LPS‐induced cells sorted onto a filter. With liquid scintillation counting we demonstrate good linearity of the c‐fos quantitation over the range of 1,000 cells to 60,000 cells per filter target. The technique is easily adapted to any sorting flow cytometer and should prove useful to help correlate any flow cytometric cell phenotype with specific mRNA abundance.
Correlation of DNA content, p53, T antigen, and V antigen in simian virus 40‐infected human diploid cells
Authors: Laffin Judith , Fogleman David , Lehman John M.
Published: August 2005

Human diploid fibroblasts (HDF) have a finite life span in cell culture which can be extended when transformed with simian virus 40 (SV40). Flow cytometric analysis of SV40‐HDF transformation allowed DNA content changes to be correlated with the appearance, quantity, and distribution of T antigen, p53, and V antigen, three proteins associated with this process. These studies demonstrated a shift in the DNA content to tetraploidy, which was correlated with the age of the SV40‐HDF but not the time of infection. A significant increase of the epitope recognized by Pab122 to host p53 and the epitope PAb101 to SV40 T antigen occurred at the same time the tetraploid population appeared. However, an antigen reactive with SV40 V antibody was present at high levels in most of the population early after infection, but the levels declined with time. The percentage of PAb101‐T antigen‐positive cells increased more rapidly in cells infected at a late passage, and this was concomitant with the shift in DNA content to tetraploid. Analysis of the mean fluorescence of total, gated populations (G1, G2, and >G2) demonstrated that a threshold level of p53 and T antigen was reached in each compartment of the cell cycle. As the transformed phenotype appeared, a population of cells was continually released into the supernatant, and although these cells had a DNA pattern similar to the monolayer cells, the T antigen and p53 levels were 3–5 times higher in the tetraploid G2 cells.

These studies correlated the expression of proteins associated with viral transformation in HDF which vary with time and shift in DNA content.
Discrimination of benign versus malignant mixed tumors of the salivary gland using digital image analysis
Authors: Layfield Lester J. , Hall Thomas L. , Fu Yao S.
Published: August 2005

Seven benign and four malignant mixed tumors of the salivary gland, biopsied using fine‐needle aspiration, were analyzed using digital image analysis. Mean nuclear form factor, perimeter, and area were significantly increased in malignant cases. Better separation between diagnostic categories, however, was achieved by utilizing the coefficient of variation (CV) within a case rather than mean value. Form factor CV alone divided cases into nonoverlapping diagnostic categories. This quantitative analog of “pleomorphism” provided a useful marker for malignancy in mixed tumors.
Continuous bromodeoxyuridine labeling and bivariate ethidium bromide/hoechst flow cytometry in cell kinetics
Authors: Poot Martin , Schmitt Heidi , Seyschab Helga , Koehler Julia , Chen Una , Kaempf U. , Kubbies Manfred , Schindler Detlev , Rabinovitch Peter S. , Hoehn Holger
Published: August 2005

Most techniques of flow cytometric cell cycle analysis are not capable of distinguishing the number of rounds of DNA synthesis that a cell has undergone since the start of an experiment. Continuous labeling with 5‐bromodeoxyuridine (Brd‐Urd) offers such a potential. We illustrate here that the bivariate analysis of non‐BrdUrd‐quenched ethidium bromide vs. BrdUrd‐quenched Hoechst 33258 fluorescence offers a high degree of resolution that enhances the analytical power of the technique, and that this approach can be applied to the analysis of a broad range of human and murine primary cells and established cell lines.
On fully automatic feature measurement for banded chromosome classification
Authors: Piper Jim , Granum Erik
Published: August 2005

Procedures for fully automatic location of chromosome axis and centromere in metaphase chromosomes are described for a practical interactive chromosome analysis system that omits the usual stages of interactive axis and centromere correction. Accuracy of centromere finding and consequential determination of a chromosome's polarity, i.e., which end is which, is measured experimentally. The saving in interaction by not correcting centromeres is compared to the increase in errors at the classification stage and the consequent increase in interaction needed to correct these errors. Some previously unreported features for banded chromosome classification are described, and in particular a set of global shape features is introduced. The discrimination capability of the feature measurements is evaluated by use of simple statistics and by reference to the performance of classifiers trained with various feature subsets. Class discrimination capability of the global shape feature set is shown to be comparable to that of centromere position, a widely used local shape feature. The variability of feature measurements that might occur in data from different laboratories on account of differing tissue, preparation methods, and digitiser hardware is assessed using three data bases of G‐banded human metaphase cells. It is shown that the differences can be considerable and that appropriate feature selection and classifier training substantially improve classification performance.
A simple electronic volume cell sorter for clonogenicity assays
Authors: Freyer James P. , Wilder Mark E. , Schor Patricia L. , Coulter James , Raju Mudundi R.
Published: August 2005

A single‐parameter electronic volume flow cell sorter that can be easily and inexpensively constructed using existing technology is described. The instrument is designed for ease and flexibility of operation, including such features as a large open area for recovering sorted cells into a variety of dishes or vessels; a remote, electrically activated fluidics system; a mechanism for heating or cooling samples during sorting; a simple arrangement for monitoring and adjusting the sorting control parameters; and an interface to a standard IBM personal computer for data acquisition, analysis, and control of the sorting windows. Several researchers in our laboratory now routinely use this sorter for plating precise numbers of cells directly into culture dishes in an aseptic manner for clonogenicity assays. The instrument can sort cells at rates of up to ∼2,000 per second with >80% sorting efficiency and no cytotoxicity. An advantage of this system is that the sorting windows can be set to exclude acellular debris and include either the entire cell volume distribution or a subset thereof. Applications of the instrument are detailed, including (1) precise cell plating for low‐dose survival studies, (2) separation of cells into age compartments, and (3) rapid inoculation of single cells into multiwell dishes for cloning studies. Advantages of this technology for cell survival studies are detailed, along with some limitations to its applicability.