bluuebunny/crossref_metadata_2025 huggingface dataset:
In situ flow cytometric analysis of nonyl acridine orange‐stained mitochondria from splenocytes
Authors: Ratinaud Marie Héaleègne , Leprat Patrick , Julien Raymond
Published: August 2005

Due to its spectral characteristics, the fluorochrome nonyl acridine orange (NAO) (λabs:489 nm, λem:525 nm), which is spontaneously incorporated by mitochondria with a high relative specificity, provides a new probe for the in situ study of these organelles by flow cytometry. In 15 min at 20°C, the dye at 4.75 × 10−6 M saturates the mitochondrial binding sites present in 1.5 × 106 cells. Unlike Rh 123, the fixation of the probe is not affected by the action of uncouplers and ionophores. Unlike acridine orange, its binding is not sensitive to nucleases.

By studying the mitochondrial incorporation of the fluorochrome during the cell cycle of murine splenocytes, it was possible to show that the biogenesis of NAO‐stained mitochondrial constituents mainly occurs during the G1 phase.
Application of orthogonal light scattering for routine screening of lymphocyte samples
Authors: Terstappen L. W. M. M. , De Grooth B. G. , Greve J. , VanBerkel W. , TenNapel C. H. H.
Published: August 2005

Orthogonal and forward light‐scattering properties of lymphocytes were measured from patients with different lymphocytic diseases in order to determine the potential value of light scattering as a screening device. Monitoring of orthogonal light scattering of lymphocytes of a B‐cell chronic lymphocytic leukemia patient during splenic irradiation (SI) revealed the selective decrease of malignant cells and the fact that the major part of the residual lymphocytes were cytotoxic lymphocytes. By combining forward and orthogonal light scattering it was shown that lymphocytes from a patient with Tλ lymphocytosis were abnormal. Orthogonal light scattering also showed an increase in cytotoxic lymphocytes in a patient with mononucleosis infectiosa and in a splenectomized patient.

Orthogonal light scattering of lymphocyte subpopulations showed that the leu8+ population of a patient with mononucleosis infectiosa was bidisperse. For elderly donors the occurrence of CD3+, CD4+, CD8+, and HNK‐1+ lymphocytes with a large orthogonal light scattering varied considerably. The CD8+ lymphocytes of these donors consisted mainly of cytotoxic lymphocytes.

These results show that determination of light‐scattering properties of lymphocytes may yield important diagnostic information and can indicate when further investigation of the lymphocytes by means of immunofluorescence is necessary.
Electrophoretic mobility studies on doxorubicin‐resistant and ‐sensitive murine P338 leukemic cells
Authors: Nair Swagata , Horton Allan , Leif Robert C. , Krishan Awtar
Published: August 2005

The electrokinetic properties of doxorubicin (DOX)‐resistant (P388/R) and ‐sensitive (P388/S) murine leukemic cells were studied in a free‐flow electrophoresis (FFE) system. The electrophoretic mobilities (EM) of P388/S and P388/R cells were 1.07 and 1.35 × 10−4cm2V−1S−1, respectively, suggesting a higher net negative charge on the P388/R cells. Neuraminidase treatment decreased the EM of both the P388/S and P388/R cells by 15–20% but had no effect on cellular doxorubicin retention. Total and cell surface sialic acid contents were similar in both the cell lines. Our studies show that no direct correlations may exist among surface charge, cell surface sialic acid content, and doxorubicin retention in DOX‐resistant and ‐sensitive P388 cells; however, differences in cell surface charge between these cell types were used to separate them by preparative FFE.
Evaluation of four methods of DNA distribution data analysis based on bromodeoxyuridine/DNA bivariate data
Authors: Lacombe F. , Belloc F. , Bernard P. , Boisseau M. R.
Published: August 2005

Four published methods of DNA‐content histogram analysis (those of Fried, Dean and Jett, simplified Dean, and Fox) were compared using a double labeling of different cell populations. Partially synchronized and asynchronous cell populations were incubated with bromodeoxyuridine (BrdUrd) and then stained with an anti‐BrdUrd monoclonal antibody and propidium iodide (PI). The fractions of cells in the G1, S, and G2 + M phases were calculated by each method and compared with those derived from G1, S, and G2 + M areas plotted on BrdUrd/DNA bivariate histograms, taken as the “true” values. This procedure enabled an optimal choice of method for a given cell population.
Practical considerations for the selection and use of optical filters in flow cytometry
Authors: Kelley Keith A. , McDowell Joan L.
Published: August 2005

The selection of proper optical filters for various excitation and emission requirements is critical in flow cytometry. Problems which arise in the selection and utilization of optical filters, and the solutions to these problems, are the subject of this article.
Different quiescence states of three culture cell lines detected by acridine orange staining of cellular RNA
Authors: Baisch Heinz
Published: August 2005

Three cell lines (CHO, L‐929 and R1H) were investigated for their growth kinetics and the difference of exponential and quiescent state of monolayers in medium with and without serum (L‐929). The noncycling populations of L‐929 and R1H in medium with serum contained increased G1‐phase percentages but also considerable proportions of SQ and G2Q cells. Although about 90% of the cells excluded trypan blue, the viability tested by colony assay was clearly lower than for exponentially growing cultures. CHO cells showed similar fractions of cells in G1‐, S‐, and G2‐Q compartments but in addition considerable cell loss. The RNA content of these cells was reduced in plateau phase by 7–48% depending on cell type and residence time in the noncycling state. The data suggest that the cells suffered from nutrition depletion and were arrested in all phases of the cycle. In contrast, L‐929 cells in medium without serum reduced their RNA content down to one‐third that of proliferating cells and still retained the full viability as shown by the same plating efficiency in a colony assay. Since about 90% of the cells had G1 DNA content, these cells resemble true G1Q or G0 cells controlled by growth factors rather than nutritional depletion.
Bromodeoxyuridine amplifies the inhibitory effect of oxygen on cell proliferation
Authors: Poot Martin , Schindler Detlev , Kubbies Manfred , Hoehn Holger , Rabinovitch Peter S.
Published: August 2005

The BrdUrd‐Hoechst method was used to analyze the interaction of various oxygen concentrations with BrdUrd substituted DNA with respect to cellular proliferation. At oxygen concentrations above 5%, human diploid fibroblast‐like cells and amniotic fluid fibroblast‐like cells showed reduced proliferation rates, which resulted from an increase in noncycling cells and from a permanent arrest of cells in the G2 phase of the cell cycle. At 35% oxygen the increase in noncyling cell fraction and the permanent arrest in G2 was strongly dependent upon the concentration of BrdUrd. Incorporation of BrdUrd into DNA, therefore, amplifies the adverse effects of increasing oxygen concentrations upon cell proliferation. The mechanism of this amplification might involve a free radical attack on DNA similar to the radiation sensitizing effect of BrdUrd.
Microtubule and microfilament distribution and tubulin content in the cell cycle of indian muntjac cells
Authors: Duanshun Wang , Suwen Li , Changqing Zeng , Ruxuan Cheng , Shaobai Xue
Published: August 2005

Using DAPI, rabbit antitubulin antibody, FITC‐labeled goat anti‐rabbit IgG, and TRITC‐phalloidin to stain individual cells, the microspectrophotometric analysis showed that three markers that represent the nucleus, microtubules (MT), and microfilaments (MF), respectively, could be recognized in individual cells without interference. The phase of the cell cycle was determined by DNA content. We found that in Indian muntjac (IM) cells, the amount of tubulin in G2 and M phases was about twice as much as that in G1 phase. In G2 cells, the cytoplasmic microtubule complex (CMTC) became denser than in G1 cells. The cytoplasmic MT extent in basically the same orientation as MF bundles in interphase. The regions where the MT is denser also have a denser MF distribution.
Histological grading, DNA content, cell proliferation and survival of patients with astroglial tumors
Authors: Zaprianov Z. , Christov K.
Published: August 2005

Light microscopy, image cytometry (ICM), and flow cytometry (FCM) were used to study the degree of differentiation, DNA content, and S‐phase of astrocytomas and glioblastoma multiforme in 102 patients. The postoperative real survival time (RST) was also studied.

Using ICM, 62 astrocytomas were investigated. Grade I astrocytomas were composed of DNA‐diploid cell lines, while grade III and glioblastoma multiforme consisted predominantly of DNA‐aneuploid lines. Moderately differentiated astrocytomas were divided as follows: 14 DNA‐diploid and 18 DNA‐aneuploid. Forty astrocytomas were studied by FCM. Using the DNA index (DI) value, cases with abnormal DNA cell lines were established in all astrocytomas, with their number increasing in grades II and III astrocytomas. FCM indicated the same subdivision of moderately differentiated astrocytomas: 12 with DNA‐diploid and 12 with DNA‐aneuploid stem lines. Patients with DNA‐diploid cell lines in the astrocytomas and low S‐fraction survived longer than patients with abnormal DNA cell populations and higher S‐fraction.

The results from this study indicate that, together with the degree of differentiation of astroglial tumors, the appearance of cell lines with abnormal DNA value and higher S‐fractions also have prognostic value.
Measurement of cell motility and morphology with an automated microscope system
Authors: Thurston Gavin , Jaggi Bruno , Palcic Branko
Published: August 2005

A method is presented which provides quantitative descriptions of the morphology and motility behaviour of a large number of unperturbed individual cells. For that, a microscope system has been developed that performs automated tracking of motile mammalian cells in tissue culture and the concurrent measurement of cell morphology. Experiments using 3T3 mouse fibroblasts show that simple global morphological features, such as degree of cell elongation, correlate with the motile behaviour of individual cells. Combined data from many cells following trypsinization and replating show the time course of the spreading of cells, measured by increase in average cell area, and the onset of cell polarization, measured by change in average cell circularity. Methods for describing fibroblast morphology and changes in morphology are discussed.
Intracellular pH measurements using flow cytometry with 1,4‐diacetoxy‐2,3‐dicyanobenzene
Authors: Cook John A. , Fox Michael H.
Published: August 2005

1,4 ‐ Diacetoxy ‐ 2,3 ‐ dicyanobenzene (ADB) has been increasingly used for measurement of intracellular pH by flow cytometry. ADB rapidly enters cells and is cleaved to the fluorescent pH indicator 2,3‐dicyano‐hydroquinone (DCH). We have analyzed several potential problems that can affect its usefulness as a pH indicator. Hydrolysis of ADB in aqueous solutions reveals the temporary presence of a fluorescent species blue‐shifted from DCH at the same pH. The presence of this species with DCH can lead to erroneous pH measurements. Stable pH measurements with ADB depend on the incubation conditions and esterase activity. Heated cells required 20 min for stable measurements, whereas control cells required 5 to 10 min. The reproducibility of pH measurements was excellent, with a resolution of ≤ 0.05 pH units in the range of 6.4 to 8.0. Absolute calibration curves of intracellular pH using the ionophore nigericin depended on matching the intracellular K+ concentration with the buffer, but relative measurements of intracellular pH were insensitive to K+. ADB was nontoxic to Chinese hamster ovary cells at up to 20 μg/ml. However, when cells loaded with dye were passed through a UV laser beam, concentrations of dye > 5 μg/ml were highly toxic. Viable cells could be sorted on the basis of intracellular pH if ADB were used at low concentrations.
Identification of inflammatory cells in bovine milk by flow cytometry
Authors: Redelman Doug , Butler Sara , Robison Jon , Garner Duane
Published: August 2005

Cells recovered from normal or mastitic bovine milk were examined by flow cytometry. All milk samples contained particulate material that was heterogeneous in size and that produced a right‐angle light‐scatter signal equal to or greater than that produced by human or bovine neutrophils. Although this material labeled with Hoechst 33342, it produced fluorescence intensities below that of intact bovine cells, suggesting that it consisted of cell fragments. Mastitic milk additionally contained other populations of cells that were poorly resolved from the normal particulate material by size (electronic volume sensor) and right‐angle light scatter. In order to improve this resolution, the milk cells were incubated with carboxydimethylfluorescein diacetate (CMFDA) to label intact cells. When milk samples labled with CMFDA were examined by dual‐parameter analysis using green fluorescence and right‐angle light scatter, five or more populations of cells could be identified in mastitic milk. These populations included intact and degenerate neutrophils, lymphocytes, including both small and activated cells, monocytes, and large activated macrophages containing many vacuoles and phagocytosed particles. Using this procedure, all the animals in the University of Nevada‐Reno Holstein dairy herd were tested once a month for 6 months. In addition, individual animals with mastitis were examined one or more times each day during the course of the inflammatory process. In the routine screening, the flow cytometric examination detected mastitis before overt symptoms developed. In cows identified to have mastitis, the flow cytometric examination provided prognostic information regarding the success of treatments.
Localization of monoclonal B‐cell populations through the use of Komogorov‐Smirnov D‐value and reduced chi‐square contours
Authors: Bagwell C. Bruce , Lovett Edmund J. , Ault Kenneth A.
Published: August 2005

In this study three different flow cytometric analysis techniques are woven together into a single system that permits improved detection of small percentages of monoclonal B cells in a milieu of either normal blood leukocytes or bone marrow cells.

This analysis is an extension of the concept of clonal excess, which is used to detect the presence of a tumor that is a clonal expansion of B cells expressing either kappa or lambda light chains.

The technique also utilizes “multiple list‐mode processing,” which is defined in this context as the simultaneous analysis of two or more listmode files that share one or more common parameters. This type of data structure enables the segmentation of two parameter light scatter displays into regions from which numerous kappa and lambda histograms subsequently can be analyzed for their respective Komogorov‐Smirnov D‐values or R‐values (reduced chi‐square value).

The final technique makes use of a calculated parameter display system. Superimposed on the light scatter dot density plot are D‐value or R‐value contours. The contours target the location of the population that is abnormal, thus providing information for setting optimal bitmap gates for clonal excess studies, other phenotypic analyses, or cell sorting.

In experiments using model systems, the sensitivity of this assay is estimated to be between 0.25% and 2.5%. The technique's distribution information and sensitivity may prove useful for staging, treatment monitoring, and relapse detection of B‐cell leukemia and lymphoma.

This application illustrates the potential of combining multiple listmode processing and calculated parameter display to expand the effective dimensionality of listmode data.
How reproducible are flow cytometry data from paraffin‐embedded blocks?
Authors: Kute T. E. , Gregory B. , Galleshaw J. , Hopkins M. , Buss D. , Case D.
Published: August 2005

The purpose of this technical report is to determine the reproducibility of flow cytometry data for ploidy and cell cycle kinetics using paraffin‐embedded blocks of breast cancer tissue. One block from each of 39 tumors was studied in this report with each block having multiple sections analyzed independently. All of these sections gave ploidy analyses, while only 34 gave cell kinetic values. The standard deviation for the DNA index value in the multiple analysis study was less than 0.1 in all but three cases. The cell kinetic values gave larger variability, and the actual values were dependent on the method of analysis. Comparison of the variability for each method of analysis could not predict which procedure was superior. These results would indicate that ploidy is a reproducible value, while cell kinetic parameters should only be used as an indicator of proliferative activity that has been normalized to the mean or median of a large set of observations processed and analyzed by the same procedure.
A flow cytometric assay for intracellular nonprotein thiols using mercury orange
Authors: O'Connor Jose Enrique , Kimler Bruce F. , Morgan Michael C. , Tempas Kathryn J.
Published: August 2005

The level of nonprotein thiols was assayed in individual mammalian cells using flow cytometry. Previous determinations of glutathione (GSH, the most abundant nonprotein thiol in most cells) by flow cytometry were based on UV laser excitation of fluorochromes. Because of several shortcoming of UV excitation, an assay for GSH using visible light is of interest. Selective staining of nonprotein thiols with mercury orange (a mercurial compound that binds stoichiometrically to sulfhydryl groups) was obtained by restricting the staining time. By using various drugs that affect GSH levels and overall thiol levels in cells, it was shown that GSH is the primary thiol group being stained. Thus a quick, specific technique using mercury orange has been developed for the flow cytometric determination of nonprotein thiols and preferentially for GSH in individual mammalian cells.
Conversion of logarithmic channel numbers into relative linear fluorescence intensity
Authors: Schmid Ingrid , Schmid Peter , Giorgi Janis V.
Published: August 2005

We describe a simple, reproducible, and generally applicable method to assess the performance of log amplifiers by using a fluorescent sample that provides multiple peaks of different intensities. The channel differences between multiple peaks are used to evaluate the logarithmic behavior of the fluorescence signal amplifier on the flow cytometer. A calibration curve can be created to correct the channel numbers for deviations from true logarithmic behavior and then convert data into relative linear intensities. By using these linear fluorescent intensities, we compared the capacity of different antisera against HIV‐1 (human immunodeficiency virus type 1) peptides to inhibit the binding of HIV‐1 to CEM, a CD4‐positive T‐cell line. A wide range of applications for this calibration procedure can be envisioned and the method is valuable for monitoring instrument performance over time.
Fluorescence response and sensitivity determination for ATC 3000 flow cytometer
Authors: Gaucher J. C. , Grunwald D. , Frelat G.
Published: August 2005

The fluorescence emitted by labeled particles after interaction with exciting light is conditioned by laser beam geometry and by the mode of fluorescence collection and filtration. A laser elliptic focusing mode is described, and the fluorescence characteristics of the sample cell flow are calculated. Fluorescence collection and detection through optical filters were analyzed, and efficiency was calculated for the ATC 3000 flow cytometer (Odam‐Bruker, Wissembourg, France). A mathematical model is proposed for calculation of the fluorescence signal and its fluctuations. The background noise for the ATC 3000 was quantified experimentally using fluorescent microspheres of a known number of bound equivalent fluorescein isothiocyanate (FITC) molecules. These experimental measurements were found to fit the theoretical predictions, thus validating the proposed model.
Imaging system for morphometric assessment of absorption or fluorescence in stained cells
Authors: Jaggi B. , Poon S. S. S. , Macaulay C. , Palcic B.
Published: August 2005

An image acquisition and processing system has been developed for quantitative microscopy of absorption or fluorescence in stained cells. Three different light transducers are used in the system to exploit the best characteristics of these sensors for different biological measurements. A digital scanner, in the form of a linear array charge‐coupled device (CCD), acquires data with high spatial and photometric resolution. A color (RGB) camera is employed when spectral information is required for the segmentation of cellular subcomponents. An image‐intensified charged‐injection device (CID) camera provides for very low light intensity measurements, primarily for fluorescence‐labeled cells. Properties of these transducers, such as contrast transfer function, linearity, and photo‐response nonuniformity, have been measured.

Two dedicated image processing units were incorporated into the system. The front‐end processor, based on a digital signal processor, provides functions such as object detection, raw image calibration, compression, artifact removal, and filtering. The second image processor is associated with the frame memory and includes a histogram processor, a dedicated arithmetic logic unit for image processing functions, and a graphics module for onebit overlay functions. An interactive program was developed to acquire cell images and to experiment with a range of segmentation algorithms, feature extractions, and other image processing functions. The results of any image operation are displayed on the video monitor. Once a desired processing sequence is determined, the sequence may be stored to become part of a command library and can be executed thereafter as a single instruction.
Image cytometry of estrogen receptors in breast carcinomas
Authors: Cohen Olivier , Brugal GÉRard , Seigneurin Daniel , Demongeot Jacques
Published: August 2005

A significant level of estrogen receptors (ER) in breast cancer cells is an indication of tumor differentiation and suggests that a homeostatic control of cell growth may persist in these cancers. In medical practice, the Dextran‐coated charcoal assays (DCCA) are still the most frequently used test to characterize patients having ER‐positive malignant breast tumors and for whom hormonal therapy is justified. Nevertheless, this routine biochemical technique is not satisfactory because it is a broad method unsuitable for revealing receptor tissue heterogeneity. However, immunocytochemical labeling, such as the ERICA method, which involves a monoclonal antibody linked to peroxidase, is a specific reaction for this purpose but which until now was not quantitative. The present study uses an original cell preparation technique combining the PAP reaction with toluidine blue counterstain for image analysis on the SAMBA system. Special software has been developed for the quantitative analysis of immunocytochemistry in cancers. Results obtained showed a high correlation between the DCCA values and the score derived from the mean ER concentration per positive tumor cell and the labeling index. In addition, intracell and intratumor heterogeneity can be displayed according to several parameters and were shown to vary according to tumor and to antiestrogen (Tamoxifen) presurgical therapy.
Modified histogram subtraction technique for analysis of flow cytometry data
Authors: Overton W. Roy
Published: August 2005

Analysis of flow cytometry histogram data by the subjective selection of an integration window can be a tedious and time‐consuming task and is often inaccurate. A new method for automated calculation of the percent positive from immunofluorescence histograms is presented. This new method is a modification of the currently used method of channel‐by‐channel histogram subtraction. Its accuracy is compared to that of the channel‐by‐channel histogram subtraction method and to another currently used automated method, which selects an integration window by finding the channels that contain the most fluorescent 2% of a control histogram.

The new histogram subtraction method is objective, easy to use, and is more accurate than other currently used automated analysis methods. PASCAL source code is given for each method of analysis.
Cyanine dye labeling reagents containing isothiocyanate groups
Authors: Mujumdar Ratnakar B. , Ernst Lauren A. , Mujumdar Swati R. , Waggoner Alan S.
Published: August 2005

New isothiocyanate derivatives of cyanine dyes were synthesized as fluorescent covalent labeling reagents for proteins and other biomolecules. These dyes have maximum absorbance in the red and near infrared regions of the spectrum, have high extinction coefficients and have adequate quantum yields. Incorporating two alkyl sulfonate groups in the dye stuctures increases their water solubility, which is beneficial for labeling biological molecules in aqueous solution. Reactivities of proteins with these new cyanines are similar to their reactivities with fluorescein isothiocyanate. These new labeling reagents are complementary to the fluorescein and rhodamine reagents, expanding the possibilities of multicolor analyses. Sheep anti‐mouse‐IgG antibody was labeled with a pentamethine cyanine dye (CY5.8‐ITC) and used with a fluoresceinated antibody as a second reagent for detecting human T‐cell subsets by flow cytometry.
Three‐color fluorescence in situ hybridization for the simultaneous detection of multiple nucleic acid sequences
Authors: Nederlof P. M. , Robinson D. , Abuknesha R. , Wiegant J. , Hopman A. H. N. , Tanke H. J. , Raap A. K.
Published: August 2005

A method is described for visualizing three nucleic acid sequences simultaneously by in situ hybridization using a new blue immunofluorescent label, amino methyl coumarin acetic acid (AMCA), in combination with green and red fluorescing FITC and TRITC.

Three chromosome‐specific repetitive probes labeled with either amino acetyl fluorene (AAF), mercury, or biotin were hybridized simultaneously to metaphase chromosomes prepared from human blood lymphocytes or to interphase tumor nuclei. Conditions for the combined use of three immunocytochemical affinity systems as well as the optimal spectral separation of the three fluorescing labels have been determined.

Three‐color in situ hybridization was applied to the study of numerical chromosome abnormalities as occur in human solid tumors. Further applications of this method in prenatal diagnosis for the detection of aneuploidy of the most frequently involved autosomes, as well as for the quantification of gene copy number and mRNA expression, are discussed.
Automatic analysis of flow cytometric DNA histograms from irradiated mouse male germ cells
Authors: Lampariello Francesco , Mauro Francesco , And Raffaella Uccelli , Spanò Marcello
Published: August 2005

An automatic procedure for recovering the DNA content distribution of mouse irradiated testis cells from flow cytometric histograms is presented. First, a suitable mathematical model is developed, to represent the pattern of DNA content and fluorescence distribution in the sample. Then a parameter estimation procedure, based on the maximum likelihood approach, is constructed by means of an optimization technique. This procedure has been applied to a set of DNA histograms relative to different doses of 0.4‐MeV neutrons and to different time intervals after irradiation. In each case, a good agreement between the measured histograms and the corresponding fits has been obtained. The results indicate that the proposed method for the quantitative analysis of germ cell DNA histograms can be usefully applied to the study of the cytotoxic and mutagenic action of agents of toxicological interest such as ionizing radiations.
A simple preservative for flow cytometric DNA analysis
Authors: Alanen Kalle A. , Klemi Pekka J. , Taimela Simo , Joensuu Heikki
Published: August 2005

A solution containing citric acid buffered saline (CABS) and 99% ethanol (E) 1:1 was used for preserving cells for flow cytometric DNA analysis. DNA histograms obtained from fine needle biopsy aspirates and preserved in CABS+E had a similar mean coefficient of variation (CV) as was obtained from aspirates taken in CABS (3.3 vs. 3.4%) and a clearly smaller mean CV than was obtained from aspirates preserved in 50% ethanol (mean 4.8%, P < .0001). Aspirates taken in CABS more often contained a small (< 3,000) number of cells as compared with aspirates preserved either in CABS+E or ethanol (P < .0001). Since preservation of cells in CABS+E allows long‐term storage of samples and results in a decreased number of insufficient samples as compared with buffered saline and in an enhanced resolution as compared with 50% ethanol, CABS+E is recommended for preservation of cytological samples to be analyzed for DNA content with flow cytometry.
Modification of a trypsin‐detergent method for DNA flow cytometry of human epidermis
Authors: Stough Dowling B. , Burns E. Robert , Mallory Susan B. , Pipkin James L. , Hinson William G.
Published: August 2005

An existing technique (Vindelov et al. Cytometry 3:323, 1983) has been modified for DNA flow cytometry of human epidermis obtained from 2 to 3 mm punch biopsies. By varying the length of time of digestion of the epidermal disc by trypsin from 5 to 70 min a controlled release of keratinocytes occurred beginning with the stratum basale and proceeding toward, but not including, the superficial layer of the epidermis, the stratum corneum.
Efficient use of monoclonal antibodies for immunofluorescence
Authors: Edwards Bruce S. , Shopp George M.
Published: August 2005

We describe here a simple and rapid small volume microplate‐based immunofluorescence staining method in which fluorochrome‐conjugated monoclonal antibodies (MAb) from three different manufacturers, used at a single standardized quantity (50 ng per test), resulted in optimal staining of human lymphocyte subsets. Staining reactions were robust, in that the number of lymphocytes used could be varied over a wide range (3 × 104‐1 × l06 cells per microplate well) without significant effects on the fluorescence intensity of staining or nonspecific binding by MAb. A measure of the efficiency of MAb use was the number of tests theoretically possible to perform with nominal 100 test kits; this figure ranged from 400 to 20,000 tests, depending on the MAb in question. This method was readily adaptable to both single‐and two‐color immunofluorescence analysis.
Detection by flow cytometry of protoplast fusion and transient expression of transferred heterologous CD4 sequences in COS‐7 cells
Authors: Rice Glenn C. , Pennica Diane
Published: August 2005

Transfer and expression of a plasmid containing the gene encoding the human T‐cell antigen CD4 by protoplast fusion was measured by flow cytometry (FCM). Protoplasts were prelabeled with fluorescein isothiocyanate (FITC) and fused to COS‐7 cells. Nonspecific protoplast adsorption to the plasma membrane was differentiated from successful protoplast fusion by the addition of an antibody directed against fluorescein to quench extracellular protoplast fluorescence. Transfection efficiencies were defined as both percent CD4 expressing cells and CD4 expression levels on a single cell basis in the transient immunofluorescence assay. Cell sorting studies indicated that intracellular protoplast‐associated fluorescence immediately after fusion exhibited a good correlation with transient CD4 transfection efficiencies as measured by indirect immunofluorescence. Reconstruction experiments comparing CD4 transfer efficiencies of protoplast fusion and calcium phosphate transfection showed that fusion resulted in a higher percentage of CD4 expressing tranfectants, while calcium phosphate transfection yielded higher CD4 expression levels on a single cell basis. Thus, FCM appears to be useful as a new tool for sensitive detection of transient expression of heterologous reporter genes in COS‐7 cell.
Chromosome banding analysis by slit‐scan flow cytometry
Authors: Bartholdi Marty F. , Meyne Julie , Johnston Roger G. , Cram L. Scott
Published: August 2005

We have investigated the use of fluorescence banding patterns for the resolution of metaphase chromosomes by slit‐scan flow cytometry. Fluorescence scans of R‐banded chromosomes have been obtained for the entire human karyotype. Metaphase chromosomes were R‐banded in suspension by staining with chromomycin A3 after hypotonic treatment in Ohnuki's buffer. Specific fluorescent landmark bands were detected for human chromosomes 1–12. Scans obtained for chromosomes 13–22 did not contain sufficient information for classification. Characteristic fluorescence patterns for human chromosomes 1 and 3 provided the clearest evidence for the detection of R‐bands by slit‐scan flow cytometry. Specific patterns were detected for human chromosomes 9–12 in which the number and placement of the fluorescent bands served as classifiers.
Vita blue: A new 633‐nm excitable fluorescent dye for cell analysis
Authors: Lee Linda G. , Berry Gillian M. , Chen Chia‐Huei
Published: August 2005

Several new derivatives of fluorescein were synthesized. The dyes were characterized by NMR; and the absorbance, excitation, and emission spectra were measured. The fluorescence quantum yields of the dyes were determinde. The pKa3 values of the dyes were measured by fluorescence titration. The characteristics of the fluorescein and sulfonefluorescein derivatives were compared. The most promising dye for use in cell analysis appeared to be compound 9, which was given the name Vita Blue. The dibutyrate ester of Vita Blue was made and the compound was given the name Vita Blue dibutyrate (VBDB, 14). The Km of VBDB with pig liver esterase was measured and found to be 4 × 10−5 M. The pKa3 of Vita Blue was 7.56 ± 0.03; both acidic and basic forms were fluorescent (dual fluorescence). The use of VBDB as an intramolecular esterase substrate and its utility for the discrimination between live and dead cells by flow cytometry is described.
Fluorescein isothiocyanate staining intensity as a probe of hyperthermia‐induced changes in chromatin conformation
Authors: Dyson J. E. D. , Britten R. A. , Battersby I. , Surrey C. R.
Published: August 2005

In a previous report we presented evidence for large increases in fluorescein isothiocyanate (FITC) fluorescent intensity caused by hyperthermia which were not associated with synthesis of heat‐shock proteins. We have now refined and considerably extended the measurements of increases in FITC fluorescent intensity caused by hyperthermia within the range 41.0°C to 50.0deg;C, and associated these with the extent of cell death caused by the hyperthermia. It appears that cell death ensues when the FITC fluorescent intensity has not returned to its baseline value within the time of one cell cycle. If thermotolerance is induced, there is a concomitant reduction in the increase in FITC staining intensity and the extent of cell death. When hyperthermia is followed by acid extraction, an additional increase in FITC staining intensity (above that due to hyperthermia alone) is observed, indicating separate sites of action on basic nuclear proteins. Hyperthermia and acid extraction have related effects on the relationship between FITC and propidium iodide staining. Hyperthermia‐induced increases in FITC staining intensity are almost completely reversed by 6.7 mM formaldehyde with a marginal effect on the control FITC staining at this formaldehyde concentration. We suggest that hyperthermia causes extensive dissociation of basic protein‐protein binding within nuclear chromatin, and that this may be a contributory cause of hyperthermia‐in‐duced cell death.
Flow cytometric studies of the nuclear matrix
Authors: Wright William D. , Higashikubo Ryuji , Roti Roti Joseph L.
Published: August 2005

We have devised a method to measure the protein and nucleic acid content of the nuclear matrix using flow cytometry. Nuclear matrices were prepared from nuclei by DNase I digestion followed by 3 M NaCl extraction. The resulting particles were stained with fluorescein isothiocyanate (FITC) for protein and propidium iodide (PI) for double‐stranded nucleic acids, and fluorescence as well as forward angle light scatter was detected. The matrices were also subjected to additional chemical or enzymatic perturbations, and changes in the above parameters were measured. Results showed that matrices from heat‐shocked cells not onlyretained the majority of heat‐induced excess nuclear protein, but also exhibited higher PI signals than controls after RNase A digestion. This observation did not hold if RNase A digestion preceded high‐salt extraction, suggesting that a salt‐extractable moiety had been replaced or altered by heat so that double‐stranded RNA was protected from the nucleolytic attack. The residual PI fluorescence in matrices from heated cells bore a linear relationship to the increased protein content in those matrices, indicating that the excess protein sequesters matrix‐associated RNA. Polyacrylamide gel electrophoresis of matrix polypeptides revealed increased amounts of many proteins as a result of heat as well as the appearance of several new proteins, one of which comigrates with the HSP72/73 heat‐shock proteins. The results of these studies show that flow cytometry can be used to study the nuclear matrix and is capable of detecting changes that result from alterations in its protein composition.
Intratumoral variations in dna distribution patterns in mammary adenocarcinomas
Authors: Askensten Ursula G. , Von Rosen Anette K. , Nilsson Roland S. , Auer Gert U.
Published: August 2005

Correlated flow‐cytometric (FCM) and microspectrophotometric (MSP) techniques were applied to investigating whether intratumoral variations in the DNA distribution patterns of 21 primary mammary adenocarcinomas can occur. Although neoplastic cell populations with both diploid and tetraploid (i.e., euploid) distribution patterns could be found in varying proportions in some of the tumors, there was no evidence in any tumor nodule for the presence of euploid populations in one part and aneuploid populations in another. This statement was based on the results of the MSP technique, where the assessments were made on cytodiagnostically identified neoplastic cells. Also, when applying the FCM technique the statement was found to be essentially valid; only one of the tumor nodules showed a DNA distribution pattern that, by means of the criteria used in this procedure, was defined as being both euploid and aneuploid. Here, however, the technique consists of assessments made on a great number of microscopically non‐identified cells.

It was concluded that when conflicting reports are given from different laboratories on the prognostic value of the cytochemically assessed DNA distribution patterns in breast carcinomas, they are not likely to be attributed to intratumoral DNA heterogeneity but, rather, to differences in the methods used and in the criteria applied for the so‐called ploidy assessments.
Initial evaluation of a new epithelial antigen (T16) for bivariate flow cytometry of bladder irrigation specimens
Authors: Bretton P. R. , Myc A. , Cordon‐Cardo C. , DeAngelis P. , Fair W. R. , Melamed M. R.
Published: August 2005

Bivariate flow cytometry (FCM) was used to study immunofluorescent T16 mouse monoclonal antibody (Mab) binding simultaneously with propidium iodide DNA measurements in bladder irrigation specimens from 30 patients with a history of bladder cancer. Aliquots of the same samples were stained with acridine orange (AO) and examined by conventional FCM. T16 Mab is believed to be specific for epithelial cells in this type of specimen and stained from 13% of the cells in a patient with cystitis to 95% of the cells in a patient with an atypical papilloma. In combination with DNA measurements, this antibody increased the sensitivity of FCM in patients with severe cystitis and relatively small numbers of tumor cells, but the diagnostic specificity may be decreased and the criteria established for interpreting univariate flow cytometry may have to be re‐evaluated and modified.
DNA flow cytometry in sperm cells from unilaterally orchiectomized patients with testicular cancer before further treatment
Authors: Fosså Sophie D. , Melvik Jan E. , Juul Niels O. , Pettersen Erik O. , Theodorsen Liv
Published: August 2005

Light microscopic sperm cell analysis and DNA flow cytometry in the seminal fluid were done in 85 testicular cancer patients after orchiectomy before further treatment. The results were compared with those from 26 healthy age‐matched males (control group). A computer‐based method for analysis of the DNA histograms was developed for evaluation of the percentage of sperm cells within the sub‐haploid, haploid (1c), and diploid (2c) and > 2c levels. Compared with the control group, testicular cancer patients had a reduced sperm cell density and sperm cell motility. The mobility grade was also significantly reduced as compared with healthy males. In addition, the number of condensed haploid sperm cells (within the subhaploid level) was decreased in testicular cancer patients, whereas the percentages of noncondensed haploid (1c), diploid, and > 2c cells were increased. Most of the DNA flow cytometric parameters were significantly correlated with sperm cell density. DNA flow cytometry in human seminal fluid offers a possible means of assessing spermatogenesis, thus providing an objective method for studying fertility disturbances, for example, in cancer patients before and after treatment.
Differences in the flow and absorption cytometric DNA distributions of mouse hepatocytes and tumor cells
Authors: Bose Kallol K. , Curley Steven , Smith William J. , Allison David C.
Published: August 2005

We have determined the DNA content, the ploidy levels, and the percentages of different cell types present in small and large mouse mammary tumors as well as in young and old mouse livers by using absorption and flow cytometry. Absorption cytometry data indicated a significant increase in the proportion of transformed G0/G1 cells in the tumors as compared to that of the stromal G0/G1 cells with progressive tumor growth. This increase was not detected by flow cytometry. In both young and old mouse livers, a small number of cells of higher ploidy (8C and 16C) were detected by absorption cytometry but were not apparent in histograms obatined by flow cytometry. Furthermore, changes in the proportions of liver cells of different ploidy with age were apparent in absorption cytometry data but not in flow cytometry data. In one mouse liver experiment, a 6C cell peak appeared in the flow cytometry histogram, but a direct measurement of DNA content by absorption cytometry failed to detect cells with such a peak. We therefore believe that some caution may be warranted in the use of flow cytometry alone for evaluation of DNA distributions and of the proportions of different types of cells in complex solid tissues.
Problem of size dependence in fluorescence DNA cytometry
Authors: Hamada Shinshichi , Fujita Setsuya
Published: August 2005

The relationship among fluorescence intensity, area, thickness, and DNA concentration of DNA‐bearing polyacrylamide film, stained with 50 ng/ml DAPI, was studied using an epi‐illumination cytofluorometer. Measurements and theoretical analysis suggest the inevitability of the size‐dependent effect on human cell nuclei in which the order of the DNA concentration is approximately 10 mg/ml, and that this effect can be virtually negated if the nuclei are stretched and flattened to less than 0.33 μm in thickness on the smears. Alternative practical ways to minimize this effect are to decrease the dye concentration of a staining solution and to make smears as thin and uniform as possible by some means such as hypotonic treatment and/or using an automatic centrifuge smearing apparatus. The size‐dependent effect, nevertheless, should be taken into account particularly when determining the DNA content of different cell types and the DNA aneuploidy of tumor cells, regardless of the technique used for measurement.
Measurement of S‐phase fractions in lymphoid tissue comparing fresh versus paraffin‐embedded tissue and 4′,6′ ‐diamidino‐2 phenylindole dihydrochloride versus propidium iodide staining
Authors: Camplejohn R. S. , Macartney J. C. , Morris R. W.
Published: August 2005

S‐phase fractions for 62 lymphoid biopsies were calculated, by means of flow cytometry, from both fresh and paraffin‐embedded tissue. The purposes of this study were to determine whether significant differences were seen between S‐phase estimates obtained from fresh and fixed tissue and to compare results obtained with two DNA dyes, namely 4′‐6′‐diamidino‐2 phenylindole dihydrochloride (DAPI) and propidium iodide (PI). The 62 cases consisted of 38 cases of non‐Hodgkin's lymphoma (NHL), 19 reactive samples, and 5 cases of Hodgkin's disease. Fifty‐four of the samples showed DNA diploid profiles. A good agreement between S‐phase results from fresh and fixed tissue was seen, with technical factors accounting for around 20% of the total variance. Using a paired t test, no significant difference was seen between fresh and fixed tissue for diploid cases, but there was a trend for S‐phase estimates from fixed tissue to be higher. If all cases (including the eight DNA aneuploid samples) were included in the analysis this difference just reached statistical significance (p <.05). In a subgroup of 19 of the cases, a comparison was performed on both fresh and fixed tissue of staining with DAPI and PI. A good agreement between results with both DNA stains was found on fresh and fixed tissue, with no significant differences being apparent, and stain‐related factors accounted for only 10% of the total variance.
Comprehensive quality assessment approach for flow cytometric immunophenotyping of human lymphocytes
Authors: Edwards Bruce S. , Altobelli Kathleen K. , Nolla Hector A. , Harper Donna A. , Hoffman Robyn R.
Published: August 2005

Flow cytometric immunophenotypic (IPT) evaluation has become an important adjunct to clinical patient management and epidemiological studies. This has precipitated a need for stringent quality assessment (QA) procedures to ascertain data integrity. We evaluated a QA approach to monitor all elements of the immunophenotyping process, inclusive of blood collection and processing procedures as well as of staining reagent and instrument performance. Central to our approach was preparation each day, in parallel with clinical analytes, of lymphocytes from healthy donors, selected from a 15 donor panel. IPT parameters evaluated over a 19 month period included frequencies of CD3+, CD4+, CD8+, and CD20+ lymphocytes and the ratio of CD4+ to CD8+ lymphocytes. The sensitivity for analytical error detection was reflected by median coefficients of variation of these parameters within individual panel donors, which were 4.1%, 4.5%, 3.9%, 8.2%, and 10.1%, respectively. IPT parameter values were determined each day for two of the panel donors, then averaged and standardized to obtain a quality or Q variate, which was the basis of QA. Error detection sensitivity decreased 0.6–1.7% and the number of false rejections increased 1.2–3.3% when one panel donor rather than two was used daily for QA. This study also illuminated important aspects of what constitutes the norm for longitudinal IPT parameter variation in healthy individuals including: (1) a generally low degree of temporal parameter variation within individual donors, but (2) significant differences between donors with respect to variance estimates for CD3+ and CD8+ lymphocyte frequencies and CD4+/CD8+ lymphocyte ratios, and (3) an apparent seasonal pattern of variation in CD4+ T‐cell frequencies.