bluuebunny/crossref_metadata_2025_split huggingface dataset:
Collodion membrane secures cells sorted by flow cytofluorometry onto microscope slides
Authors: Meck Robert A. , Benson Neal A. , Ng Alan G. , Price Brandon J. , Ingram Marylou 
Published: August 2005

AbstractQuantitative microscopic cytology of cells previously sorted by flow cytofluorometry has been hindered by the loss of cells from the microscope slide during staining procedures. The simple application of a semipermeable membrane of collodion over fixed or unfixed cells sorted directly onto a microscope slide secured virtually 100% of the cells onto the slide. Cells covered with the collodion membrane stained with Papanicolaou's stain as well as routine clinical cervical cytologic preparations. In contrast, fewer than one half the cells sorted onto uncoated or albumin coated slides were retained after staining.
Analytical cytometric approaches to heterogeneous cell populations in solid tumors: A review
Authors: Dethlefsen Lyle A. , Bauer Kenneth D. , Riley Reba M. 
Published: August 2005

AbstractThe problems encountered in studying the heterogeneity of cells in solid tumors is reviewed with emphasis on the role of various analytical cytometric assays for studying both the biology and the dynamics of proliferating, quiescent and dead malignant cells in vitro and in vivo. Due to advances in cytometric technology, many interesting in vitro studies on tumor cell heterogeneity have been and will be conducted over the next several years. For example, the acidic acridine orange staining of HeLa cells in suspension culture does readily discriminate between proliferating and quiescent cells. Some of these assays have been and others will be extended to in vivo studies. However, it is obvious that either the current analytical cytometric techniques must be modified and refined to permit better resolution for the complex situation in vivo or other new analytical cytometric techniques will have to be developed before many interesting studies on tumor cell heterogeneity in vivo can be addressed with reasonable efficiency.
New cell cycle compartments identified by multiparameter flow cytometry
Authors: Darzynkiewicz Zbigniew , Traganos Frank , Melamed Myron R. 
Published: August 2005

AbstractSimultaneous measurements of cellular DNA and RNA as well as estimates of the sensitivity of DNA in situ to denaturation by acid (which correlates with the degree of chromatin condensation) and cell ability to incorporate 5‐bromodeoxyuridine (BUdR), performed on over 40 different cell systems enabled us to subclassify cells into 12 functionally distinct cell cycle compartments. Quiescent cells had low RNA values, DNA very sensitive to denaturation, and they did not incorporate BUdR. Although in most cell systems they had 2C DNA content (G1Q), quiescent cells with higher DNA values (SQ and G2Q) were also seen. The G1 phase of exponentially growing cells had two distinct compartments, A and B. G1 cells entered S phase only from the B compartment. An increase in RNA and a decrease in the sensitivity of DNA to acid denaturation beyond a specific level characterized the transition of cells from the A to B compartments. Since the G1A to G1B transition was not linear but exponential, it may be assumed that a non‐deterministic event triggering cell progression into the cycle resides within G1A. Under adverse growth conditions the probability of a G1A to G1B transition decreased. Cells In G2 had DNA more sensitive to acid denaturation than S or G1 phase cells. Mitotic cells had the most condensed chromatin and their RNA content was twice that of G1A cells. Differentiated cells were characterized by 2C DNA (G1D) but, depending on the cell type, had varying RNA content and different degrees of chromatin condensation. A hitherto undescribed category of cells undergoing transition from quiescence to the cycle (or vice versa) was distinguished based on their intermediate values of RNA, sensitivity of DNA to acid denaturation and inability to incorporate BUdR at the rate characteristic for S cells. Depending on their DNA content (C) at the time of transition these cells could be classified as G1T (2C), ST (4>C>2) or G2T (4C).
Evaluation of the S phase distribution of flow cytometric DNA histograms by autoradiography and computer algorithms
Authors: Sheck L. E. , Muirhead K. A. , Horan P. K. 
Published: August 2005

AbstractCell sorting and tritiated thymidine autoradiography were used to define the distribution of S phase cells in flow cytometric DNA histograms obtained from exponential mouse lymphoma cells (L5178Y). The numbers of labeled S phase cells, autoradiorgraphically determined from cells sorted at 2‐channel intervals in the G1/early S and late S/G2M regions of the histogram, were compared with the numbers of computed S phase cells in comparable 2‐channel intervals as predicted by several computer algorithms used to extract cell cycle phase distributions from DNA histograms. Polynomial and multirectangle algorithms gave computed estimates of total %S in close agreement with the tritiated thymidine labeling index for the cell population, while multi‐Gaussian algorithms underestimated %S. Interval autoradiographic and algorithm studies confirmed these results in that no significant differences were found between the autoradiographic S phase distribution and S phase distributions calculated by the polynomial and multirectangle models. However, S phase cells were significantly underestimated in G1/early S by a constrained multi‐Gaussian model and in both G1/early S and late S/G2 by an unconstrained multi‐Gaussian model. For the particular cell line (L5178Y), staining protocol (mithramycin following ethanol fixation) and instrumentation (Coulter TPS‐2 cell sorter) used in this study, close agreement between computed %S and tritiated thymidine labeling index was found to be a reliable indicator of an algorithm's success in resolving S phase cells in the G1/S and S/G2 transition regions of the DNA histograms.
A fluorescence polarization flow cytometer
Authors: Keene J. P. , Hodgson B. W. 
Published: August 2005

AbstractThe effect of mitogens on the degree of polarization of the fluorescence from viable human lymphocytes, labeled with fluorescein, have been reported to be different in cells from subjects with malignant disease as compared with those from healthy donors. This has potential applications in the field of clinical diagnosis and a flow cytometer could be the instrument of choice for this purpose. We believe that such an instrument should be simple and easy to use and a flow cytometer was assembled with this in mind and with the object of gaining experience which would provide the basis for a future finalized design. The instrument is suitable for use in other biologic applications where polarization is of interest and also as a general purpose flow cytometer. Its main features, its characteristics and a preliminary performance report are presented here.
A dual laser flow sorter utilizing a CW pumped dye laser
Authors: Arndt‐Jovin Donna J. , Grimwade Brian G. , Jovin Thomas M. 
Published: August 2005

AbstractA dual laser flow sorter has been constructed from an existing single laser system by incorporating a dye laser as the second laser source. The argon ion laser emission was used both as a pump laser and as a source by beam splitting before entrance to the dye laser. The emissions of the dye laser and pump laser beams were recombined and focused with the same optical train used in the single laser system. The imaging on the stream of the flow sorter provided for spatial and thus temporal separation of the exciting beams and subsequent sample emissions.
Simultaneous quantitation of hoechst 33342 and immunofluorescence on viable cells using a fluorescence activated cell sorter
Authors: Loken Michael R. 
Published: August 2005

AbstractThe cytochemical stain Hoechst 33342 has been used to quantify DNA in viable cells and has been used under nonsaturating conditions to discriminate between lymphoid cell types. In order to correlate the quantitative emission from Hoechst 33342 with cell surface antigens, a fluorescence activated cell sorter was modified to simultaneusly detect emission from the UV excited Hoechst dy and fluorescein attached to the cell surface by immunofluorescence techniques. A special set of laser mmirrors was installed in an argon ion laser so that all the lines from 351‐488 nm could be used to illuminate the cells. Appropriate emission filters were used to separate the light emitted by Hoechst 33342 from the fluorescein. An electronic cross‐over circuit was used to compensate for special overlap between the two dyes. Analysis of murine lymph node cells stained both with Hoechst 33342 under nonsaturating conditions and anti‐Thy 1.2 indicated that the cells that stained dimly with the Hoechst dy expressed the Thy 1.2 marker while the cells that were brightly stained with Hoechst 33342 lacked this differentiation antigen. The correlation of cell surface myeloma protein with cell cycle on an in vitro cell line indicated that the heterogeneity of cell surface antigen expression could not be accounted for solely by variations occurring during the cell cycle.
Enzyme kinetic studies in cell populations using fluorogenic substrates and flow cytometric techniques
Authors: Watson James V. 
Published: August 2005

AbstractTwo flow cytometers, an Ortho Cytofluorograf model 4800A and a “custom” built dual laser instrument, have been interfaced with a PDP 11/40 computer to assay the rate of accumulation of fluorescent products in populations of single cells due to enzymatic hydrolysis of fluorogenic substrates. Esterase activity assayed with fluorescein diacetate exhibited abnormal Michaelis‐Menten kinetic behavior in EMT6 cells. This was in marked contrast to normal kinetics obtained with homogenates assayed with a conventional spectrofluorimeter. Moreover, the homogenates were 4 5x less efficient at converting substrate than were intact cells. Fluorescein diacetate is lipophilic and the induced fluorescence was evenly distributed throughout intact cells. In contrast a second substrate studied, 3‐O‐methyl fluorescein phosphate, is lipophobic and highly polar and the induced fluorescence was located at the cell surface. Results obtained with this substrate suggested that membrane, but not intracellular phosphatases were being assayed. Although the reaction product appeared to be lost rapidly from the site of hydrolysis it was possible to extract Km and Vmax for the dephosphorylation which followed normal kinetics. The dual laser flow cytometer was built to study 4‐methylum‐belliferone based substrates (UV excitation) simultaneously with those based on fluorescein. An assay using fluorescein diacetate and 4‐methylumbelliferyl acetate simultaneously was able to discriminate between a mouse mammary tumour (EMT6) and a human colonic adenocarcinoma cell line (HT 29). Furthermore, incubation of both cell lines for up to 6 hr with these substrates maintained viability at about 90%.
DNA measurements of chromosomes 9 and 22 of six patients with t(9;22) and chronic myeloid leukemia
Authors: Geraedts J. P. M. , Van der Ploeg M. 
Published: August 2005

AbstractMore than 90% of the cases of chronic myeloid leukemia are characterized by the presence of a so‐called Philadelphia chromosome. This Philadelphia chromosome has been shown to be a product of a translocation between chromosomes 9 and 22 in the majority of cases. It is present in the bone marrow and unstimulated blood cultures. Visual examination of this chromosome gave the impression that small differences in length might exist between patients. Therefore, DNA measurements were undertaken on six patients with t(9;22) and chronic myeloid leukemia. Metaphases obtained from unstimulated peripheral blood cultures were Q‐banded for photography and identification of chromosomes 9 and 22. Feulgen staining was performed, and the preidentified cells were photographed again. Cytophotometry of the photographic negatives was done using a Hidacsys‐Arrayscan computer program. Relative DNA contents were calculated for the normal chromosomes 9 and 22 and both chromosomes involved in the translocation. Between 7 and 17 cells were measured per patient. The results indicated that no differences existed between the patients regarding the amount of material translocated from chromosome 22 to 9. In all patients the sum of the DNA contents of the normal chromosomes 9 and 22 was less than that of the translocation chromosomes. Possible explanations for this observation are discussed.
A new flow cytometric pulse height analyzer offering microprocessor controlled data acquisition and statistical analysis
Authors: Kachel V. , Schneider H. , Schedler K. 
Published: August 2005

AbstractThe instrument described is capable of storing up to 112 one parameter histograms (128 channels) or 16 one parameter and 3 two parameter histograms (64 × 64 channels). A low cost 10 Mhz oscilloscope displays the graphical and alphanumerical data. During data acquisition, the original pulses, an analogue rate meter and the growing histogram are displayed simultaneously on the screen. The instrument may be completely operated by an 18 element keyboard. The data processor allows calculation of integrals, mean values, standard deviations and coefficients of variation of operator selectable regions of the acquired histograms. Gaussian distributions determined by the statistical parameters may be compared directly with the measured distributions. Distribution curves can be normalized, shifted, added, subtracted or compared with artificial distributions which are generated by superimposition of distinct Gaussian curves. Exponentially fitted background curves can be subtracted from the measured histograms. A text line at the top of the screen displays the numerical values and explanatory remarks. The two parameter histograms may be rotated, observed from the sides or back and projected either on the x‐or y‐axes. The projections or selected lines or columns can be transferred to the one parameter range and treated statistically. Sequences of one parameter distributions may be transferred to the two parameter range and displayed as pseudo‐two‐parameter histograms.
The effect of isoproterenol on nuclear protein synthesis in electrostatically sorted rat hepatocytes
Authors: Pipkin J. L. , Hinson W. G. , Hudson J. L. , Martin J. L. , North B. M. , Pack L. D. 
Published: August 2005

AbstractRat hepatocyte nuclei were electrostatically sorted and collected from specific regions of the cell cycle. Proteins were extracted from homogeneous populations of nuclei with cetyltrimethylammonium bromide and chromatographed by one‐ and two‐dimensional acid‐urea polyacrylamide gel electrophoresis. Small quantitative differences were observed in amido black stained two‐dimensional patterns of the nuclear proteins of the G0 + G1 and G2 + M populations. However, comparison of the two‐dimensional autoradiographs of 3H‐leucine labeled nuclear proteins in these populations revealed dramatic differences. After isoproterenol administration, animals showed enhanced labeling of certain nuclear proteins in G2 + M populations, but no noticeable effect in protein labeling was observed in the G0 + G1 populations. Four days after the administration of isoproterenol, fluorescent histograms exhibited increased amounts of DNA in the G2 + M populations. Comparison of the total amino acid composition of two proteins from control animals revealed the distinct chemical contrast of these proteins. Both proteins possessed higher ratios of acidic to basic residues than anticipated for basic proteins. These procedures and findings should prove useful for further understanding of mechanisms of toxicant effects upon nuclear protein metabolism.
High resolution optics combined with high spatial reproducibility in flow
Authors: Eisert W. G. 
Published: August 2005

AbstractAccurate sizing in flow using optical methods generally requires high resolution optics and specially designed flow systems. Flow systems developed by this group have following features: (a) double sheath configuration for optical index match, (b) no curved optical surface in the sensing area, (c) gradual hydrodynamic focusing over a long distance to minimize mechanical shearing, (d) precision spatial positioning of cells by reducing suspension fluid diameter to a cell diameter or less, (e) total thickness between outer surfaces of the flow chamber at the viewing area of 1.5 mm or less.Cells intersect a laser light beam focussed to circular as well as elliptical cross‐sections of 1μ or less in diameter. Cellular extinction is monitored during transit through the beam. Cell length is derived from the time for flight measurement and corrected for absolute values by continuous velocity reference using a second laser beam intersecting the cell stream at a predetermined distance. This second spot may be circular or elliptical, of a different polarization and/or frequency.Simultaneous fluorescence intensity and diameter measurements were performed on test particles using different optical geometries. The influence of the particle structure on fluorescence measurements is demonstrated where high resolution sizing is required at the same time.
A morphometric expression of differentiation in fine‐needle biopsies of breast cancer
Authors: Stenkvist Björn , Bengtsson Ewert , Eriksson Olle , Jarkrans Torsten , Nordin Bo 
Published: August 2005

AbstractAn automated method of particle sizing in fine‐needle biopsies of human breast carcinomas is described. The average size of clusters of cells aspirated from breast carcinomas was strongly correlated with cytologic differentiation of the tumors and also with the recurrence rate 3 years after mastectomy so that smaller cluster size was associated with poor differentiation and a poor prognostic outcome. It thus seems possible to express in quantitative terms the degree of differentiation of breast carcinomas.
Evaluation of selected aerosol‐control measures on flow sorters
Authors: Merrill John T. 
Published: August 2005

AbstractFlow sorters produce microdroplets as part of their normal operation, and if these microdroplets escape into the room, they are potentially hazardous. We have tested several aerosol‐control measures on a commercial flow sorter. To accomplish this, T‐4 phages were introduced into the sorter's liquid jet through the sample injection tube, and culture plates containing lawns of T‐4‐sensitive Escherichia coli bacteria were exposed around the sorter for each operational configuration. After the exposure, the plates were incubated and then scored for plaques. A single phage‐containing microdroplet landing on a plate was sufficient to cause a plaque of lysed bacteria to form. The number of plaques was thus an indicator of how much aerosol was released for each configuration. Aerosols were controlled most effectively by catching the central, undeflected stream in a vacuum‐exhausted tube; this technique, coupled with the manufacturer's vacuum‐exhaustion of the air around the sorting location, produced no plaques. Several failure modes were tested, including having the central stream hit the outside of the catch‐tube or deflection plates, and loss of the manufacturer's vacuum‐exhaustion system. Many flow sorters allow the undeflected stream to splash into a flask or beaker, an approach that produces the most plaques if instrument‐failure modes are excluded. The simple addition of a catch‐tube removed this major contributor to the aerosol production.
A flow system for partial automation of plating efficiency tests
Authors: Macevicz S. , Dean P. , Peters D. , Pinkel D. , Gray J. 
Published: August 2005

AbstractA flow system that sterilely deposits a known number of cells into a collection vessel has been constructed and evaluated. In this device, the cells from a single‐cell suspension flow through a nozzle that constrains them to the center of the emerging jet. The cells in the jet are illuminated by a 0.5‐mW helium‐neon laser; they are detected, sized and counted on the basis of the light scattered as they pass through the laser beam. The device has been used for plating efficiency tests in a tissue culture laboratory. We found that it saved considerable labor by eliminating the dilution step from the standard plating efficiency procedure. The biologic effect of machine plating was determined by comparing plating efficiencies of KHT, CHO‐T5, and CHO‐AA8 cells established by hand plating with those established by machine plating. Results indicate that machine plating reduces the plating efficiency of the CHO‐T5 cells while the plating efficiencies of the CHO‐AA8 and KHT cells are unaffected.
Cytophotometric analysis of lytically and persistently infected tissue culture cells with measles virus
Authors: Harms H. , Aus H. M. , ter Meulen V. 
Published: August 2005

AbstractLytically and persistently infected VERO and LUB cells were cytophotometrically characterized with the aid of computer analysis. Images scanned at 260 and 280 nm were processed by computer algorithms. With this approach, infected cells could be segmented and differentiated from uninfected cells. Lytically and persistently infected cells could be distinguished by distinct differences in nuclear and cytoplasmic optical densities. These findings are supported by biologic data based on the analysis of virus‐specific proteins and nucleic acids. The applied computer aided cytophotometry provides a new approach in the study of virus‐cell interaction.
Automated selection of metaphase cells by quality,
Authors: van den Berg H. T. C. M. , de France H. F. , Habbema J. D. F. , Raatgever J. W. 
Published: August 2005

AbstractThe performance of metaphase‐finding systems could be improved if they were able to determine the quality of the cells detected. This paper discusses the extent to which this may be realized by the introduction of a metaphase‐quality parameter. Data obtained from 300 cells were statistically analyzed. Seventeen features were measured and nine visual properties were determined for each cell. Discriminant analysis and regression analysis were used to extract those features and visual properties which contribute to assessment of metaphase quality. Rather low correlations were found between the selected measured features and visual properties. A quality‐parameter based on a linear combination of cluster projections, areas and perimeters was found to account for 64% of the variation between visual and measured quality indicators. In addition, an increase in the predictive value for finding usable metaphases from 28–68% was achieved.
Cell cycle dependency of tumor promoter on murine erythroleukemic cells,
Authors: Zucker Robert M. 
Published: August 2005

AbstractThe tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) affected the cell cycle of murine erythroleukemic cells. In logarithmically growing cells a transient G1 block lasting approximately 6 hr developed after the addition of TPA. To investigate this cell cycle effect of TPA, cells were synchronized either by a double thymidine block or by centrifugal elutriation and were analyzed by flow cytometry and electronic nuclear volume. Using double thymidine blocked synchronized cells or elutriated separated fractions containing cells other than G1, a transient G1 block occurred as the cells entered early G1. The G1 elutriated cell population was blocked in the G1 period by TPA before cell cycle traverse. This transient block in G1 was not observed after the next cell cycle traverse. Addition of TPA to butyric acid or dimethylsulfoxide induced‐cells resulted in a transient G1 block in addition to inhibition of differentiation. TPA was more inhibiting on dimethylsulfoxide‐induced cells than butyric acid‐cells.