bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcein: A novel marker for lymphocytes which enter lymph nodes
Authors: Weston Susan A. , Parish Christopher R.
Published: August 2005

Previous studies have identified unique cell surface antigens which are associated with the specific binding of lymphocytes to high endothelial venules (HEV). Evidence is presented in this paper which demonstrates that uptake of the fluorescent dye calcein by lymphocytes represents au additional marker for the lymph node homing subpopulation of lymphocytes. Calcein exhibits a characteristic ability to label lymphocytes differentially into two distinct populations, based on fluorescence intensity, that does not occur with three other structurally related, fluorescein‐based dyes. In vivo lymphocyte migration studies revealed that cells displaying the “dull” fluorescence phenotype, although entering all lymphoid organs examined, preferentially homed to the lymph nodes, particularly the popliteal lymph node (PLN). By contrast, lymphocytes displaying the “bright” phenotype were essentially excluded from entering lymphoid organs, where entry is HEV dependent, but were observed entering spleen, where entry is HEV independent. Furthermore, a high proportion (76. 5%) of lymphocytes displaying the dull fluorescence phenotype expressed the PLN homing receptor MEL‐14. Based on these observations it is suggested that calcein uptake may be a marker for general membrane properties, such as fluidity and plasticity, essential for the passage of lymphocytes through HEV. © 1992 Wiley‐Liss, Inc.
Automated image detection and segmentation in blood smears
Authors: Poon Steven S. S. , Ward Rabab K. , Palcic Branko
Published: August 2005

A simple technique which automatically detects and then segments nucleated cells in Wright's giemsa‐stained blood smears is presented. Our method differs from others in (1) the simplicity of our algorithms; (2) inclusion of touching (as well as nontouching) cells; and (3) use of these algorithms to segment as well as to detect nucleated cells employing conventionally prepared smears. Our method involves: (1) acquisition of spectral images; (2) preprocessing the acquired images; (3) detection of single and touching cells in the scene; (4) segmentation of the cells into nuclear and cytoplasmic regions; and (5) postprocessing of the segmented regions. The first two steps of this algorithm are employed to obtain high‐quality images, to remove random noise, and to correct aberration and shading effects. Spectral information of the image is used in step 3 to segment the nucleated cells from the rest of the scene. Using the initial cell masks, nucleated cells which are just touching are detected and separated. Simple features are then extracted and conditions applied such that single nucleated cells are finally selected. In step 4, the intensity variations of the cells are then used to segment the nucleus from the cytoplasm. The success rate in segmenting the nucleated cells is between 81 and 93%. The major errors in segmentation of the nucleus and the cytoplasm in the recognized nucleated cells are 3.5% and 2.2%, respectively. © 1992 Wiley‐Liss, Inc.
Fluorescence ratio measurements of double‐labeled probes for multiple in situ hybridization by digital imaging microscopy
Authors: Nederlof P. M. , van der Flier S. , Vrolijk J. , Tanke H. J. , Raap A. K.
Published: August 2005

To expand the multiplicity of the in situ hybridization (ISH) procedure, which is presently limited by the number of fluorochromes spectrally separable in the microscope, a digital fluorescence ratio method is proposed

For this purpose, chromosome‐specific repetitive probes were double‐labeled with two haptens and hybridized to interphase nuclei of human peripheral blood lymphocytes. The haptens were immunocytochemically detected with specific antibodies conjugated with the fluorochromes FITC or TRITC. The FITC and TRITC fluorescence intensities of spots obtained with different doublehaptenized probes were measured, and the fluorescence ratio was calculated for each ISH spot. Combinations of different haptens, such as biotin, digoxigenin, fluorescein, sulfonate, acetyl amino fluorene (AAF), and mercury (Hg) were used. The fluorescence intensity ratio (FITC/ TRITC) of the ISH spots was fairly constant for all combinations used, with coefficients of variation between 10 and 30%

To study the feasibility of a probe identification procedure on the basis of probe hapten ratios, one probe was double‐labeled with different ratios, by varying the relative concentrations of the modified nucleotides (biotin‐11‐dUTP and digoxigenin‐11‐dUTP) in the nick‐translation reaction

Measurement of the FITC and TRITC intensities of the ISH spots showed that the concentration of modified nucleotides used in the labeling procedures was reflected in the mean fluorescence intensity of the ISH spots. Furthermore, the ratio distributions showed little overlap due to the relatively small coefficients of variation

The results indicate that a multiple ISH procedure based on fluorescence ratio imaging of double‐labeled probes is feasible. © 1992 Wiley‐Liss, Inc.
Quantification of fluorescence in situ hybridization signals by image cytometry
Authors: Nederlof P. M. , van der Flier S. , Verwoerd N. P. , Vrolijk J. , Raap A. K. , Tanke H. J.
Published: August 2005

In this study we aimed at the development of a cytometric system for quantification of specific DNA sequences using fluorescence in situ hybridisation (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome‐specific probes were labeled with biotin, digoxigenin, or fluorescein. The instrumentation requirements are evaluated

Quantification of the fluorescence ISH signals was performed using an epi‐fluorescence microscope with a multi‐wavelength illuminator, equipped with a cooled charge couple devices (CCD) camera. The performance of the system was evaluated using fluorescing beads and a homogeneously fluorescing specimen

Specific image analysis programs were developed for the automated segmentation and analysis of the images provided by ISH. Non‐uniform background fluorescence of the nuclei introduces problems in the image analysis segmentation procedures. Different procedures were tested. Up to 95% of the hybridization signals could be correctly segmented using digital filtering techniques (min‐max filter) to estimate local background intensities

The choice of the objective lens used for the collection of images was found to be extremely important. High magnification objectives with high numerical aperture, which are frequently used for visualization of fluorescence, are not optimal, since they do not have a sufficient depth of field

The system described was used for quantification of ISH signals and allowed accurate measurement of fluorescence spot intensities, as well as of fluorescence ratios obtained with double‐labeled probes. © 1992 Wiley‐Liss, Inc.
Immunoblotting technique to study release of melanophore‐stimulating hormone from individual melanotrope cells of the intermediate lobe of Xenopus laevis
Authors: de Rijk E. P. C. T. , Terlou M. , Cruijsen P. M. J. M. , Jenks B. G. , Roubos E. W.
Published: August 2005

The melanotrope cells in the pays intermedia in the pituitary of Xenopus laevis synthesize and release the melanophorestimulating hormone (αMSH), a small peptide that causes skin darkening during the process of background adaptation. Evidence has been found for a heterogeneity in biosynthetic activity of the melanotrope cells. In the present study two questions were addressed: (1) does the melanotrope cell population also show heterogeneous αMSH‐release, and (2) can this heterogeneity be changed by extracellular messengers? Since dopamine is known to inhibit αMSH‐release, this messenger is used to study the regulation of the heterogeneity. To quantify αMSH‐release from individual cells, a cell blotting procedure has been developed for the binding and relative quantification of the small αMSH peptide. The immunoblotting procedure involves binding of the cells to a carrier slide and binding of released αMSH to a nitrocellulose filter. After immunostaining, the amount of αMSH per cell was quantitated by image analysis. Untreated melanotrope cells reveal a distinct variability in αMSH‐release, some cells showing low secretory activity, whereas others are strongly secreting, indicating heterogeneity of αMSH‐release. Dopamine treatment strongly inhibits αMSH‐release from individual cells, resulting in a clearly less pronounced melanotrope cell heterogeneity. The effect of dopamine appears to be dose‐dependent as a low dopamine concentration has only a moderate effect on the αMSH‐release. It is proposed that dopamine is a physiological regulator of the degree of melanotrope cell heterogeneity in αMSH‐release. © 1992 Wiley‐Liss, Inc.
Comparison of BrdUrd and [3H]TdR incorporation to estimate cell proliferation, cell loss, and potential doubling time in tumor xenografts
Authors: Zätterström Ulf K. , Johansson Maria , Källén Anders , Baldetorp Bo , Oredsson Stina , Wennerberg Johan , Killander Dick
Published: August 2005

In this study, two different methods of estimating cell proliferation were compared: cell loss and potential growth rate of xenografted head and neck cancer grown in nude mice based on the detection of DNA incorporation of bromodeoxyuridine (BrdUrd) in one method, and [3H]thymidine ([3H]TdR) in the other. The 21‐d‐old xenografts were labelled in vivo, either with BrdUrd or [3H]TdR and excised at intervals during 65.5 h. In tumors containing BrdUrd, the percent labelling was measured in mid‐S and mid‐G1 phase windows of cytograms from bivariate DNA flow cytometry (FCM). In [3H] TdR‐labelled tumors, the percent labelled mitoses (PLM) was determined by light microscopy evaluation of autoradiographs. With a computer program based on a theoretical model, the percent labelling versus time after injection was used to analyze cell cycle time, cell loss tumor growth fraction, and potential doubling time. The values calculated from DNA incorporation with BrdUrd agreed well with those obtained from labelling with [3H]TdR, i.e., cell cycle time 2.3 vs. 2. 4 d, and growth fraction 67 vs. 70%. The estimated potential doubling time was 3.1 d and cell loss factor 40% by both methods. Flow cytometry analysis of BrdUrd‐labelling is considerably faster than the evaluation of [3H]TdR‐labelling, and the present results provide further support for the BrdUrd labelling method as a promising alternative to the PLM method in cell cycle studies designed to evaluate the relevance of cell proliferative properties in relation to biological behavior in xenografted head and neck cancer. © 1992 Wiley‐Liss, Inc.
Intercalation of anthracyclines into living cell DNA analyzed by flow cytometry
Authors: Belloc Francis , Lacombe Francis , Domain Patrice , Lopez Francis , Bernard Philippe , Boisseau Michel R. , Reifers Josy
Published: August 2005

Anthracyclines (ANT) are used in the treatment of leukemia and other cancers. These drugs have beers shown to intercalate between the strands of DNA. In the present study, we show that the amount of ANT intercalated into DNA can be determined by measuring the fluorescence resonance energy transfer (FRET) between Hoechst 33342 (H33342) and ANT bound to DNA. The transfer efficiency was found to depend on the amount of disposable ANT but was independent of the amount of H33342 bound to DNA over a wide range of H33342 concentrations. The method was adapted for flow cytometric measurement of FRET in whole living cells and was used to evaluate the degree of intercalation of daunorubicin (DAU) and idarubicine (IDA) into DAU‐sensitive and DAU‐resistant leukemic cell lines. ANT intercalation into DNA was affected by factors which modify the intracytoplasmic concentration of ANT, and it was shown that the action of ANT and the resistance to ANT could not be attributed solely to the intercalative effect of the drugs. The method has advantages over previously described methods and represents a useful complementary tool in studies on the mode of action of ANT and the mechanisms of chemoresistance. © 1992 Wiley‐Liss, Inc.
Flow cytometric titration of retroviral expression vectors: Comparison of methods for analysis of immunofluorescence histograms derived from cells expressing low antigen levels
Authors: Sladek Todd L. , Jacobberger James W.
Published: August 2005

Few quantitative studies addressing immunofluorescence histogram analysis have been published. One study by Overton (Cytometry 9:619–626, 1988) has shown threshold and histogram subtraction methods to be accurate for analysis of well‐separated immunofluorescence distributions of positive and negative cells. An evaluation of methods to analyze immunofluorescence histograms when positive and negative immunofluorescence distributions overlap has not, to our knowledge, been reported. In this paper, data obtained from flow cytometry of immunofluorescently stained cells infected with recombinant retroviruses that produce a range of simian virus 40 large T antigen levels were analyzed by threshold, histogram subtraction, and distribution modeling methods. This analysis showed that as the separation between the immunofluorescence distributions of positive and negative cell populations decrease the best methods for histogram analysis are modeling followed, in order, by histogram subtraction, and threshold analysis. © 1993 Wiley‐Liss, Inc.
Flow cytometric quantitation of nucleoside transporter sites on human leukemic cells
Authors: Jamieson Gary P. , Brocklebank Anne M. , Snook Marie B. , Sawyer William H. , Buolamwini John K. , Paterson Alan R. P. , Wiley James S.
Published: August 2005

Quantitation of equilibrative, nitrobenzylthioinosine (NBMPR) sensitive (es) nucleoside transporters on blast cells isolated from patients with acute myeloblastic leukemia is useful in predicting intracellular accumulation of the antileukemic nucleoside drug, cytosine arabinoside. We previously reported the synthesis of a fluorescein‐labeled ligand for the es nucleoside transporter, 5‐(SAENTA‐x2)‐fluorescein. This paper reports the synthesis of 5‐(SAENTA‐x8)‐fluorescein in which the linkage between fluorescein and nucleoside ligand has been increased from 2 atoms to 8 atoms. This new ligand had a sixfold increase in affinity (Kd 0.9 ± 0.1 nM) as well as an 86% increase in the cell associated fluorescence output compared to its prototype 5‐(SAENTA‐x2)‐fluorescein. The fluorescence signal arising from 5‐(SAENTA‐x8)‐fluorescein specifically bound to freshly isolated and cultured leukemic myeloblasts was converted to molecules of equivalent soluble fluorescein (MESF) using standardized fluorescein microbeads and compared with the number of es nucleoside transporter sites assayed concurrently by [3H]NBMPR equilibrium binding analysis. A high correlation between the two assays was observed (r = 0.98), which enabled the cell‐bound fluorescence output of 5‐(SAENTA‐x8)‐fluorescein to be expressed in numbers of es nucleoside transporter sites per cell. The improved properties of 5‐(SAENTA‐x8)‐fluorescein over those of its prototype molecule make it a suitable reagent for flow cytometric quantitation of nucleoside transporter expression on leukemic cells isolated from patient samples. © 1993 Wiley‐Liss, Inc.
Increased sensitivity of damaged DNA to digestion with nuclease S1 as assessed in single cells by flow cytometry
Authors: Prosperi Ennio , Supino Rosanna , Bottiroli Giovanni
Published: August 2005

DNA sensitivity to digestion with nuclease S1 was investigated in cells irradiated with gamma rays, or treated with the antitumor drug adriamycin (Adr). The nuclease‐resistant DNA fraction was determined by propidium iodide staining. Treated cells were found to be more sensitive to nuclease digestion than the undamaged controls. Gamma ray‐induced strand breaks were detectable at doses up to 10 Gy; an increase in the reaction temperature, from 37° to 63°C, was necessary in order to detect higher levels of damage. Nuclease S1 sensitivity in Adr‐treated cells showed a single‐peak, concentration‐dependent relationship, in agreement with the known self‐inhibitory effect exerted by high drug doses. Determination of DNA digestion could be performed in combination with other cellular parameters (e.g., protein content). Detection of drug‐resistant cells in a heterogeneous population of small‐cell lung carcinoma was achieved on the basis of the different sensitivity of the cells to enzymatic digestion. These results indicate that nuclease S1 may be a useful probe for studying in single cells DNA alterations induced by drugs or radiation. © 1993 Wiley‐Liss, Inc.
Rhodamine 123 fluorescence of immortal hybridoma cell lines as a function of glucose concentration
Authors: Borth Nicole , Kral Gerda , Katinger Hermann
Published: August 2005

The fluorescence of rhodamine 123 stained cells has been described to specifically reflect the activity of mitochondria. Changes in the intensity of fluorescence observed in stimulated lymphocytes were attributed to an increased glycolytic activity of cells due to increased growth rates. Previously reported changes in mitochondrial activity observed in batch cultures were likewise attributed to changed growth rates. In this study we report that the Rh123 fluorescence of hybridoma cell lines in batch culture more closely correlates to the glucose concentration in the culture supernatant than to growth rates. When cells are transferred into glutamine free medium with defined glucose concentrations ranging from 0 to 3,000 mg/L the mean Rh123 fluorescence adapts to the respective glucose concentration within 6 hours and gives a linear correlation. This can be explained by the previously described dependence of specific glucose consumption rates on glucose availability in the medium. The importance of controlling glucose availability, especially in large scale fermentations, is discussed. © 1993 Wiley‐Liss, Inc.
Neural network analysis of DNA flow cytometry histograms
Authors: Ravdin Peter M. , Clark Gary M. , Hough John J. , Owens Marilyn A. , McGuire William L.
Published: August 2005

A pattern recognition system based on Neural Network Analysis, a form of artificial intelligence, was used to search DNA flow cytometry histograms for features that correlated with breast cancer patients' risk of relapse. DNA flow cytometry histograms and clinical followup information from 796 breast cancer patients were used to train a Neural Network to predict the clinical outcome of patients in a separate independent set of 794 patients. Median follow‐up in this patient data base was short, 23 months. Neural Network Analysis resulted in a model that evaluated DNA flow cytometry histograms differently than conventional analysis, which categorizes the histograms by ploidy and S‐phase fraction. Neural Network Analysis appeared to identify low risk and high risk subsets of patients as accurately as conventional analysis. Neural Network Analysis placed heavy emphasis on the region to the right of the diploid G2/M peak, where a subpopulation of nuclei with high DNA content is seen even in many histograms scored as diploid by conventional techniques. The number of nuclei in this region was found to be a powerful predictor of patient outcome, and multivariate analysis showed that the number of nuclei in this region and the S‐phase fraction both were independently predictive of relapse. This pilot study suggests that conventional analysis (based on a mechanistic interpretation of regions in flow cytometry histograms) might be used in conjunction with and improved by pattern recognition systems or insights derived from them. © 1993 Wiley‐Liss, Inc.
Pulse modulation of the excitation light source boosts the sensitivity of an arc lamp‐based flow cytometer
Authors: Steen Harald B. , Sørensen Otto I.
Published: August 2005

It has been found that the type of short arc high‐pressure lamps used in some flow cytometers can be pulsed to reach intensities many times higher than that measured when they are operated on constant power. A 75 W mercury‐xenon lamp was fed 20 m̈s current pulses superimposed on its rated current of 5.4 A. Pulses of 50 A produced a 75‐fold increase of the emission intensity at the excitation wavelength of FITC, which means that the excitation intensity in the flow chamber at this wavelength exceeded 100 mW. At the major emission lines of mercury, i.e., 366, 436, and 546 nm, the increase was about 25‐fold, corresponding to intensities of the order of 300 mW. The light pulses were found to be reproducible to within 2% and the intensity was independent of the pulse frequency up to at least 250 pulses/s. Operated in this mode the lamp produced more than 1 · 108 30 A pulses, which is sufficient to measure some 10,000 typical size samples. The rise time of the light pulses was about 5 m̈s. This was sufficiently short that the leading edge of the light scattering signal from a cell entering the excitation focus of the flow cytometer could be used to trigger the current pulse so that the cells were exposed to the full light pulse intensity while they were still within the focus. Since the sensitivity of flow cytometers is proportional to the square root of the excitation intensity, the resulting increase of the sensitivity should correspond to a lowering of the detection limit for FITC by a factor of about 8. The actual improvement as measured by means of calibrated FITC‐labelled particles was 9.8, thus bringing the FITC sensitivity of the present arc lamp‐based flow cytometer on par with that of the best laser‐based instruments commercially available. © 1993 Wiley‐Liss, Inc.
Phase‐resolved fluorescence lifetime measurements for flow cytometry
Authors: Pinsky Bertram G. , Ladasky John J. , Lakowicz Joseph R. , Berndt Klaus , Hoffman Robert A.
Published: August 2005

A flow cytometer capable of measuring fluorescence lifetimes by the phase shift method has been built and evaluated. Under optimal conditions, the resolution of the fluorescence lifetime measurement is shown to be under 200 picoseconds. Pulse intensity variations are normalized using limiting amplifiers and electronic filtering. Normalization of signal intensities provides a lifetime measurement that is independent of fluorescence intensity over at least a 50‐fold (17 dB) range in fluorescence intensity. The fluorescence lifetimes of unbound dye, fluorescent beads, cells stained with ethidium bromide, propidium iodide, and phycoerythrin‐conjugated monoclonal antibodies have been measured. The fluorescence lifetimes measured for these particles are well correlated with lifetime measurements made using a standard fluorimeter. Cells stained with ethidium bromide and propidium iodide at various nucleotide‐to‐dye ratios are shown to exhibit similar behavior to static cuvette measurements. The fluorescence lifetime parameter is also shown to resolve phycoerthyrin fluorescence from propidium iodide fluorescence. © 1993 Wiley‐Liss, Inc.
Immunofluorescent evaluation of DNA repair synthesis using interactive laser cytometry
Authors: Srivastava Vinod , Miller Susan , Busbee David
Published: August 2005

An improved differential fluorescence analysis technique which employs scanning laser cytometry for the detection of two DNA binding fluorochromes was used to quantitate both total DNA and cellular incorporation of the base analogue, bromodeoxyuridine (BrdUrd), into UV‐ or methyl methane sulphonate (MMS)‐treated DNA. In this procedure DNA containing BrdUrd was partially denatured and immunolabeled by binding anti‐BrdUrd monoclonal IgG to incorporated BrdUrd. Anti‐BrdUrd was detected with a secondary fluorochromelabeled polyclonal anti‐IgG. Cells were counterstained with propidium iodide to allow the determination of total DNA. Fluorescence was determined using the Meridian ACAS 570 Scanning Laser Cytometer. The computer‐analyzed images were generated from monodisperse populations of UV‐ or MMS‐treated cells, allowing the evaluation of DNA synthesis associated with excision repair and of total DNA content in single cells. These data were compared with 3H‐thymidine incorporation occurring as a function of excision repair. The analysis of DNA synthesis with this technique is consistent, relatively simple, and rapid with excellent sensitivity and provides a viable method for determining cycling vs. non‐cycling cells, total cellular DNA, and excision repair‐associated DNA synthesis of individual cells within heterogeneous cell populations. © 1993 Wiley‐Liss, Inc.
Shape changes in Escherichia coli B/r A during agar filtration
Authors: Vardi E. , Grover N. B.
Published: August 2005

We have investigated the phenomenon of shape distortion in a sample of 1,552 Escherichia coli B/r A cells in balanced exponential growth, during preparation for electron microscopy by agar filtration. Mixed preparations of bacterial cells and polystyrene latex spheres were shadow cast at low angle and the resulting shadows used to obtain quantitative estimates for the dimensions of the dehydrated cell; these then serve as a basis for a model of its shape in three dimensions. A statistical analysis of the projections of clustered cells and the intervening fissures, in nonshadow‐cast preparations, provides an estimate of the effects of drying. The average width of the dehydrated cell (450 nm) is about 20 nm greater than the diameter of the live bacterium, whereas its length (1,398 nm) is ∼ 40 nm less. © 1993 Wiley‐Liss, Inc.
Flow cytometric clonal excess analysis of peripheral blood, routine handling, and pitfalls in interpretation
Authors: Johnson Anna , Olofsson Tor
Published: August 2005

Clonal excess (CE) analysis by flow cytometry is a convenient method to detect minimal involvement of peripheral blood and bone marrow by B‐cell lymphoma. The method is based on evaluation of the congruity between κ and λ light chain distributions within a normal B‐cell population. By using the Kolmogorov‐Smirnov method for evaluation of histogram identity, the maximum difference (D value) between distributions is calculated. However, variable adsorption of cytophilic plasma immunoglobulin (Ig) to CD16 positive cells, T‐cell subsets, and the B cells themselves may cause incongruity between the light chain distributions that might simulate or disguise a true clonal excess and thus create major pitfalls in the interpretation of the CE analysis. These phenomena are observed both in normal blood donors and in patients with non‐Hodgkin's lymphoma. In the present report we describe: (1) how freezing of the isolated mononuclear cells before immunostaining effectively removes most of the adsorbed cytophilic Ig, (2) how by exclusion of the CD16 positive population further resolution of the relevant histogram shape is obtained to avoid false interpretations of incongruity, and (3) how adsorption of Ig to B‐cells creates a typical pattern of “reciprocal labeling” that can be mistaken for a clonal excess. Based on our observations we argue against an uncritical use of normal D values for definition of clonal excess and advocate an analytic evaluation of the κ and λ overlay distribution by dual immunofluorescence, either by excluding CD16 positive cells or by including only B cells, to reveal the nature of the different deviations. © 1993 Wiley‐Liss, Inc.
Resolution of fluorescence signals from cells labeled with fluorochromes having different lifetimes by phase‐sensitive flow cytometry
Authors: Steinkamp John A. , Crissman Harry A.
Published: August 2005

A flow cytometric method has been developed that uses phase‐sensitive detection to separate signals from simultaneous fluorescence emissions in cells labeled with fluorochromes having different fluorescence decay lifetimes. By CHO cells were stained with propidium iodide (PI) and fluorescein isothiocyanate (FITC). These dyes bind to DNA and protein and the fluorescence lifetimes of the bound dyes are 15.0 and 3.6 ns, respectively. Cells were analyzed as they passed through a modulated (sinusoidal) laser excitation beam. Fluorescence was measured using only a long pass filter to block scattered laser excitation light and a single photomultiplier tube detector. The fluorescence detector output signals were processed by dual‐channel phase‐sensitive detection electronics and the phase‐resolved Pland FITC signals were displayed as frequency distribution histograms and bivariate plots. By shifting the phase of one detector channel reference signal by π/2 + ψ1 degrees and the phase of the other detector channel reference signal by −π/2 + ψ2 degrees, where ψ1 and ψ2 are the phase shifts associated with the PI and FITC lifetimes, the PI and FITC signals were separately resolved at their respective phase‐sensitive detector outputs. This technology is also applicable to suppressing background interferences caused by cellular autofluorescence, unbound/free dye, nonspecific dye binding, and Raman and Rayleigh scattering. © 1993 Wiley‐Liss, Inc.
Flow injection cytometry: A new approach for sample and solution handling in flow cytometry
Authors: Lindberg Walter , Ruzicka Jaromir , Christian Gary D.
Published: August 2005

A prototype instrument for sample and solution handling in flow cytometry has been constructed. The system is modular and allows the control of any combination of up to 4 pumps, 2 selection valves, and 2 injection valves. These devices are controlled by a computer using TTL‐logic. The flow injection instrument is interfaced to the flow cytometer via a 6‐port injection valve, thereby facilitating virtually any flow pattern to be implemented without affecting the fluidics of the cytometer. Salient features of the instrument are accurate control of the volume of injected sample and reagent, reproducible timing, and controlled mixing conditions. Results from model experiments of on‐line staining of trout erythrocytes with different concentrations of 4′,6‐diamidino‐2‐phenylindole (DAPI) and propidium iodide (PI) are shown. Possible ways to improve the performance and utility of the instrument are also discussed. © 1993 Wiley‐Liss, Inc.
A comparison of techniques for introducing macromolecules into living cells
Authors: Lee Grace , Delohery Thomas M. , Ronai Zeev , Brandt‐Rauf Paul W. , Pincus Matthew R. , Murphy Randall B. , Weinstein I. Bernard
Published: August 2005

Flow cytometry has been used to evaluate several techniques for introducing macromolecules into large numbers of living cells. One technique is cell fusion with red blood cell ghosts loaded with a fluorescent reporter molecule (RBCF). The second technique, termed osmotic lysis of pinosomes (OLP), involves a brief exposure of cells to a hypertonic solution containing the reporter molecule; subsequently, a hypotonic media is added which lyses the pinosomes formed during the hypertonic treatment. A third technique, scrape loading (SL), involves the creation of transient holes in the cell membrane through the application of mechanical forces, which allows for the passage of reporter molecules into cells. A comparison of these techniques is presented here. OLP appears to offer several advantages: It is a simple procedure, virtually all cells are fluorescently labelled, and it is capable of loading larger amounts of material more uniformly into cells while maintaining excellent viability. © 1993 Wiley‐Liss, Inc.
Flow cytometric screening of blood samples for malaria parasites
Authors: van Vianen Philip H. , van Engen Anneloes , Thaithong Sodsri , van der Keur Maarten , Tanke Hans J. , van der Kaay Hugo J. , Mons Barend , Janse Chris J.
Published: August 2005

An automated method for the detection and estimation of malaria parasites in blood samples using flow cytometry is presented. In a single‐step procedure 50 μl of blood sample was collected in 1 ml of lysis solution containing formaldehyde, causing red blood cells to lyse while parasites and white blood cells are preserved. Thus prepared, samples could be transported and remained stored in lysis solution until flow cytometric analysis was performed. The cells were stained for DNA with the fluorescent dye Hoechst 33258 and subsequently analyzed by a FACStar flow cytometer. Parasites and white blood cells were distinguished and counted based on blue Hoechst fluorescence and forward scattering. Since red blood cells were lysed, parasite numbers were given related to the number of white blood cells similar to what is done in microscopic examination of thick blood smears. In dilution experiments with animal and human material, parasite counts by flow cytometry correlated very well with the theoretically calculated numbers (regression coefficients of > 0.94). In human material parasitemias of ∼0.005% were detected. In a pilot Study, 700 samples were collected in Thailand and screened by microscopic examination of thick smears and by flow cytometry; 29 were found positive by combining both methods, 2 were missed by flow cytometry, and 20 were missed by microscopists in the field. After microscopic reexamination in the central laboratory, 15 of these 20 were found positive, 5 remained unconfirmed. © 1993 Wiley‐Liss, Inc.
Nationwide quality control trial on lymphocyte immunophenotyping and flow cytometer performance in Italy
Authors: Brando Bruno , Sommaruga Elisabetta
Published: August 2005

The Italian Society for Cytometry (Gruppo Italiano di Citometria, GIC) promoted this nationwide large‐scale quality control trial on cellular immunophenotyping and flow cytometer performance in 1991. The aim of this independent study was to evaluate instrument performance with calibrated fluorescence microbeads (minimum threshold for FITC, linearity, coefficients of variation and resolution indexes for fluorescence), and to correlate it to the measurement of lyophilized lymphocyte surface immunofluorescence staining with a wide spectrum of antibodies (percentage of positive cells and fluorescence mean intensity).

A single send‐out was made to 306 laboratories with 350 instruments throughout Italy. Each kit included anonymous vials containing premixed calibrated microbeads, lyophilized human lymphocytes and small aliquots of conjugated monoclonal antibodies, for both single FITC and double FITC/PE staining. Participants were also asked to use their own anti‐CD4 monoclonal. A valid answer was returned by 209 laboratories with 221 instruments. Gating was not an assay variable. The minimum sensitivity threshold for FITC ranged from 26.6 to 11,293 MESF, with marked instrument heterogeneity as far as the relationship between FITC threshold and coefficients of variation for fluorescence was concerned. A low FITC threshold also correlated with a good resolution index for low intensity stainings. No performance comparisons among instrument brands and models were made. The lyophilized lymphocyte analysis showed an overall frequency of outliers ranging from 3.2% to 19.7%, with a maximum for CD2 FITC (19.7%) and CD3 + HLA−DR+ (12.1%). A strongly negative relationship was evident between the FITC threshold and the number of CD2+ cells, whereas the sensitivity threshold had virtually no effects on the measured level of higher antigen density markers like CD4 and many others. Absolute fluorescence intensity measurements of CD4+ and CD19+ were also made. This study design proved valid and suitable for a large‐scale evaluation of both instrument performance and cytometer operators' skill. A number of practical problems were identified among participants, thus stressing the need for more effective educational programmes and technical guidelines. © 1993 Wiley‐Liss, Inc.
Flow cytometric estimation of poly(A) + RNA by fluorescent in situ hybridization
Authors: Belloc Francis , Lacombe Francis , Dumain Patrice , Mergny Jean Louis , Lopez Francis , Bernard Philippe , Reiffers Josy , Boisseau Michel R.
Published: August 2005

A method for the detection of poly(A) + RNA in cell suspensions by in situ hybridization and flow cytometry is described. The hybridizing properties of oligothymidylate o(dT) was well preserved after coupling to fluorescein. This fluorescent oligonucleotide was used as a probe to determine the poly(A) + RNA content of fixed HL60 leukemic cells by flow cytometry. Labeling was considerably reduced by treating the cells with RNAse, and by competitive hybridization with either poly(U) or free poly(A). Labeling was also decreased in a time‐dependent fashion by incubating the cells with actinomycin D prior to fixation. This method represents an improvement on the methods measuring total RNA and could be of value in investigations on the effect of drugs on RNA metabolism. © 1993 Wiley‐Liss, Inc.
Identification of a marker chromosome in a rat ascitic tumor by flow cytometry using a modified method for preparing chromosome suspensions
Authors: Manogaran P. S. , Pande Gopal
Published: August 2005

Preparation of good chromosome suspensions is a crucial step in obtaining good flow karyotypes from different types of cells. Although several methods are in use, not all are suitable for all cell types. Primary cells from murine tissues are specially difficult to flow karyotype. We have modified the existing protocols for preparing chromosome suspensions from the Zajdela ascitic hepatoma (ZAH) cells of rat and have constructed their flow karyograms. Our karyograms show a distinct marker chromosome in these cells, which could not be resolved using other protocols. We have also used this method for preparing chromosomes from the bone marrow cells of the rat and have compared their flow karyograms with the ZAH karyotype. We expect that our protocol will be useful for studying other types of tumor and normal cells. © 1993 Wiley‐Liss, INc.
DNA content measurements and an improved idiogram for the Indian muntjac
Authors: Levy Howard P. , Schultz Roger A. , Ordonez José V. , Cohen Maimon M.
Published: August 2005

The Indian muntjac, an asiatic deer, has the lowest diploid chromosome number among mammals (female 2N = 6; male 2N = 7). Using flow cytometric quantification of propidium iodide‐stained cells, we determined the DNA content of muntjac cells to be 94% that of human. This suggests that the muntjac may serve as a model for investigation of karyotypic evolution and rearrangement. In order to facilitate future comparative gene mapping studies, computer‐aided analysis of digitized metaphase chromosomes allowed development of a detailed Indian muntjac G‐banded idiogram incorporating both ISCN‐type nomenclature and quantitative estimates of the size of each band and position. © 1993 Wiley‐Liss, Inc.
Identification of pig chromosomes in pig‐mouse somatic cell hybrid bivariate flow karyotypes
Authors: Bouvet Alain , Konfortov Bernard A. , Miller Nigel G. A. , Brown David , Tucker Elizabeth M.
Published: August 2005

To identify pig chromosomes in pig‐mouse somatic cell hybrids, dual‐laser flow karyotypes and GTG‐banded metaphase spreads of pig, mouse, and 7 pig‐mouse hybrid cell lines were compared. Pig chromosomes no. 1, 2, 5, 6, 10, 11, 13, 14, 16, 18, X and Y were tentatively assigned to individual peaks in the pig flow karyotype on the basis of DNA content vs. relative chromosome length. In the 7 hybrid cell lines, 7 out of 8 peaks distinct from those of the mouse cell line could be correlated with the presence of pig chromosomes no. 5, 9, 10, 11 or 16, 14, 15, and 18, whereas 1 peak appeared to correspond to the presence of 1 middle‐size chromosome (3, 4, or 7). Other pig chromosomes present in the hybrids could not be detected with certainty due to superposition with mouse peaks and mouse chromosome rearrangements. © 1993 Wiley‐Liss, Inc.
Kinetic analysis of cytotoxic lymphocyte—target cell interaction as quantified by dual parameter flow cytometry
Authors: Waniewski Jacek , Palucka A. Karolina , Porwit Anna
Published: August 2005

The kinetics of conjugate formation between leukemic cell lines (K562 and Daudi) and lymphokine‐activated killer (LAK) cells was studied. A flow cytofluorometry method using double immunofluorescence staining was applied. During the first 15 min of incubation of LAK effectors with leukemic targets, a rapid binding occurred, followed by a plateau phase lasting until 30 min of observation. A considerable, yet not statistically significant, between‐donor variability was noticed. A mathematical model of conjugate formation kinetics, based on the analogy to enzyme kinetics, was formulated and validated. Parameters of the model were related to the binding capacity of effector and target cells, and to the lifetime of conjugates and free cells. The concordance of theoretical curves with experimental data proved that the described model can be considered as a useful tool for the evaluation of kinetic and dynamic characterization of conjugate formation between leukemic targets and LAK effectors. © 1993 Wiley‐Liss, Inc.
Quantitation of simian virus 40 T‐antigen correlated with the cell cycle of permissive and non‐permissive cells
Authors: Lehman John M. , Friedrich Thomas D. , Laffin Judith
Published: August 2005

These studies examined cell cycle progression and quantitative changes in T‐antigen following infection by SV40. Single cells were assayed by multiparameter flow cytometric analysis (FCM) for DNA content and T‐antigen expression. Conditions were used which permitted permissive, semi‐permissive, and non‐permissive cells to be monitored through two rounds of DNA synthesis induced by SV40. The permissive cells included the monkey kidney cell lines; CV‐1, Vero and BSC‐1 and the COS‐1 and COS‐7 which are CV‐1 cells transformed with an origin defective SV40. The non‐permissive cell strains included mouse embryo fibroblasts, Chinese hamster fibroblasts, and IMR‐90, a human diploid fibroblast. Cell types differed in the maximal amount of T‐antigen expressed per cell. Additionally, all cell types expressed a limited quantity of T‐antigen for each cell cycle phase and the quantity increased in each successive phase. The level in each phase was increased only two‐fold when 100 times more virus was used. Thus, for an infected population the quantity of T‐antigen was dependent on cell cycle distribution. High levels of T‐antigen were not required for permissive infection; however, permissive cells were distinguished from non‐permissive cells by the G2 levels. Permissive G2 cells had more than double the T‐antigen content expressed in G1, while nonpermissive G2 cells had less than a two‐fold increase over G1 levels. The appearance of cells with tetraploid DNA content and the failure to undergo mitosis correlated to the higher T‐antigen levels in the G2 of the permissive cells. Two other strains of SV40, 776, and VA45 exhibit similar values for T‐antigen expression and movement into tetraploid DNA content. This study establishes the levels of T‐antigen correlated to the cell cycle and cell type. © 1993 Wiley‐Liss, Inc.
Quantitative analysis of mitotic and early‐G1 cells using monoclonal antibodies against the AF‐2 protein
Authors: Di Vinci Angela , Geido Elio , Pfeffer Ulrich , Vidali Giorgio , Giaretti Walter
Published: August 2005

We have recently described a novel protein (AF‐2), conserved between fission yeast and man, and we have shown by flow cytometry (FCM) that AF‐2 is highly accessible to specific monoclonal antibodies (MoAbs) in mitotic and postmitotic early‐G1 phase cells. The aim of the present study was to optimize the FCM methodology using MoAbs against AF‐2 and to show that the evaluation of the mitotic cells, using different cell lines, was quantitative and reproducible. We found that a method based on fixation with ethanol, instead of formalin, resulted in improved DNA histogram coefficients of variation and implemented separation of early‐G1 cells from late‐G1 cells. In addition, by eliminating several cell permeabilization and protein salt extraction steps, the method became straightforward, conserved a clear‐cut separation of the green fluorescence of M‐ with respect to G2‐phase cells, and did not significantly affect cellular integrity. The coefficient of correlation among the mitotic index values evaluated by this FCM method using MoAbs against AF‐2 and by microscopic visual counting was R=0.94. When the FCM/AF‐2 method was tested against an independent FCM method, which allows clear separation of M‐ and G2‐phase cells according to 90° scattering, we found R=0.93. We conclude that MoAbs against the AF‐2 protein may be used in FCM for quantitative analysis and for isolation of M‐phase cells, providing as well, the identification of the early‐Gl cell subcompartment. The method may, in addition, be useful for the simultaneous detection of cytoplasmic cytokeratin and nuclear AF‐2 antigen. © 1993 Wiley‐Liss, Inc.
Flow cytometric DNA content and clinical outcome in patients with non‐small cell lung cancer given postoperative radiation therapy
Authors: Yu Jinming , Shaeffer James , Zhu An , Kuban Deborah A. , El‐Mahdi Anas M. , Philput Christine B.
Published: August 2005

DNA content by flow cytometry was assessed in 47 cases from a series of 130 patients with non‐small cell lung carcinoma (NSCLC) given radiation therapy postoperatively. This was done in an attempt to identify which patients might benefit, or not benefit, from postoperative radiotherapy. From archival formalin‐fixed paraffin‐embedded specimens, 16 of the 47 cases (34%) had DNA diploid tumors while 31 cases (66%) were nondiploid. A diploid DNA content was significantly related to improved overall survival (P = 0.0061) and tumor‐free survival (P = 0.0167) but not with frequency of tumor recurrence within the irradiated field. Histological grade was not significantly related to overall survival, tumor‐free survival, or frequency of in‐field tumor recurrence. DNA content was found in this study of NSCLC patients irradiated postoperatively to be a useful marker for predicting survival but not for predicting local recurrence. © 1993 Wiley‐Liss, Inc.
Whole blood method for simultaneous detection of surface and cytoplasmic antigens by flow cytometry
Authors: Landay Alan , Jennings Cheryl , Forman Meryl , Raynor Robert
Published: August 2005

Dual parameter analysis of surface antigens in flow cytometry has become a standard method for detection of cell subsets. However, few methods have been described for the extension of multiparameter analyses to include cytoplasmic or intracellular antigens. Here we describe a simple and reproducible method for simultaneous detection of surface and intracellular antigens by flow cytometry in lysed whole blood samples. This method employs the use of digitonin, a mild glycoside detergent, and formaldehyde for permeabilization and fixation. Red blood cells are lysed with 2% acetic acid. Preparation of samples in this manner resulted in altered light scatter characteristics relative to unpermeabilized samples; however, gating issues were overcome using a combination of scatter vs. fluorescence gating. Quantitation of CD3+/CD4+ and CD3+/CD8+ cells using this method was equivalent to counts obtained with the reference method using a commercially available lysis procedure and fluorescence vs. scatter gating. The effectiveness of the permeabilization process was assessed using a monoclonal antibody designated TIA‐1, which is specific for a cytoplasmic antigen associated with cytotoxic granules predominantly found in CD8+ cells. The method effectively quantitated TIA‐1 positive cells and demonstrated the specificity of the reagent for a subpopulation of CD8+ lymphocytes. Using this simplified procedure for simultaneous identification of surface and cytoplasmic antigens could help in studies of cell activation, proliferation, and other functional characteristics of the immune system. © 1993 Wiley‐Liss, Inc.
Two‐Color flow cytometric analysis of intraerythrocytic malaria parasite DNA and surface membrane‐associated antigen in erythrocytes infected with Plasmodium falciparum
Authors: Pattanapanyasat Kovit , Udomsangpetch Rachanee , Webster H. Kyle
Published: August 2005

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Separation of rooster spermatogenic nuclei by means of centrifugal elutriation
Authors: Boix Jacint , Roca Joaquim
Published: August 2005

By application of minor variations in the usual method of centrifugal elutriation, we have been able to separate a nuclear suspension from rooster testes. We have obtained a population of elongated or late spermatid nuclei (98% purity), a population of round or early spermatid nuclei (90% purity), and, finally, a mixed population of meiotic and pre‐meiotic nuclei. © 1993 Wiley‐Liss, Inc.