bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flow cytometric double labeling technique for screening of multidrug resistance
Authors: Gheuens Eric E. O. , van Bockstaele Dirk R. , van der Keur Maarten , Tanke Hans J. , van Oosterom Allan T. , De Bruijn Ernst A. 
Published: August 2005

AbstractWe investigated the capabilities of flow cytometry in the analysis of a multidrug resistant (MDR) human ovarian cancer cell line 2780AD and its drug sensitive parental A2780. A functional assay using daunorubicin (DNR) as a fluorescent probe was combined with an immunofluorescence assay of P‐glycoprotein (P‐gp) using the monoclonal antibody MRK‐16. Functionally MDR could be demonstrated by the lower DNR‐content of MDR cells compared to DNR‐content of drug sensitive cells. When incubation was performed with DNR in the presence of verapamil, DNR‐content increased in the MDR cells. However the content of the A2780 cells was never attained. Differences in DNR‐content were not related to differences in DNA‐content. In experimental cell lines immunofluorescence data were inversely related with those of DNR‐content: MDR cells had high levels of P‐gp expression and low levels of DNR‐content (and vice versa in drug sensitive cells). Both assays can be easily combined in a multiparametric flow cytometric procedure to evaluate both parameters simultaneously in the same cells. Analysis of clinical samples demonstrates the existence of aberrant subpopulations which would not be detected by using a single parameter assay.
Measuring potential doubling times of murine tumors using flow cytometry
Authors: Carlton Jeffrey C. , Terry Nicholas H. A. , White R. Allen 
Published: August 2005

AbstractFlow cytometric analysis of bromodeoxyuridine (BrdUrd) labeled cells has led to the description of various methods for determining population potential doubling times (Tpot) from samples obtained at a single time point. There have been several reports of results from human tumor samples using these various methods of analysis, but little documentation of the validity of these approaches in experimental tumor models. Recently, we reported results using in vitro cell cultures and determined the most reliable method for these. In this paper we report the results of this methodology when applied to two in vivo murine tumor models (MCaK and FSA). The analytic approach tested is summarized as follows: The relative movement (RMlu(t)), a measure of the mean DNA fluorescence of BrdUrd‐labeled cells that have not undergone division at the time of sampling, and a quantity ν, a function of the fractions of BrdUrd‐labeled divided and undivided cells, were measured for a series of time points following labeling. From each value of RMlu(t) an estimate of the length of S‐phase (Ts) was computed and from each ν and Ts a value for Tpot was found. These results were compared to the values of Ts and Tpot obtained by fitting all the values simultaneously. Tumor MCaK values of Ts and Tpot of 9.6 and 28.0 h were obtained from fitting all of the data. Tumor FSA gave values of 16.8 and 42.3 h for Ts and Tpot, respectively. The results of this analysis show that single time point measurements can give reliable estimates of Ts and Tpot.
Recombinant glutathione S‐transferase (GST) expressing cells purified by flow cytometry on the basis of a GST‐catalyzed intracellular conjugation of glutathione to monochlorobimane
Authors: Puchalski Ralph B. , Manoharan T. Herbert , Lathrop Angela L. , Fahl William E. 
Published: August 2005

AbstractCOS cells transiently expressing glutathione S‐transferase (GST) π, Ya, or Yb1 (human Pi, rat Alpha or Mu, cytosolic classes) were purified by flow cytometry and used in colony‐forming assays to show that GST confers cellular resistance to the carcinogen benzo[a]pyrene (±)‐anti‐diol epoxide (anti‐BPDE). We developed a sorting technique to viably separate recombinant GST+ cells (20%) from the nonexpressing electroporated population (80%) on the basis of a GST‐catalyzed intracellular conjugation of glutathione to the fluorescent labeling reagent monochlorobimane (mClB). The concentration of mClB, length of time cells are exposed to mClB, and activity of the expressed GST isozyme determined the degree to which recombinant GST+ cells fluoresced more intensely than controls. On‐line reagent addition ensured that all cells were exposed to 25 μM mClB for 30–35 s during transit before being analyzed for fluorescence intensity and sorted. The apparent Km for mClB of the endogenous COS cell GST‐catalyzed intracellular reaction was 88 μM. Stained GST Ya+ or Yb1+ cells catalyzed the conjugation 2 or 5 times more effectively than GST π+ cells. Enzyme activity in cytosolic fractions prepared from sorted recombinant GST+ cells was 1.8 ± 0.3‐fold greater than that of the control (80 ± 4 nmol/min/mg protein). Upon a 5‐fold purification of GST π+ cells in the electroporated population, resistance to anti‐BPDE in colony‐forming assays increased 5 times, from 1.1‐fold (unsorted) to 1.5‐fold (sorted) (P <0.001).
Simultaneous flow cytometric method to measure phagocytosis and oxidative products by neutrophils
Authors: Perticarari S. , Presani G. , Mangiarotti M. A. , Banfi E. 
Published: August 2005

AbstractWe developed a rapid and sensitive two‐color flow cytomotric method which allows the simultaneous quantification of both the phagocytosis rate and the oxidative burst activation of polymorphonuclear leukocytes (PMNLs). The oxidation of hydroethidine (HE) to ethidium bromide (EB) was performed by the oxidative neutrophil products within the cells during the respiratory burst, which was stimulated by phagocytized fluorescein‐labeled Staphylococcus aureus. By means of flow cytometry we measured red EB fluorescence emission together with green fluorescence, which was emitted by the ingested fluoresceinated bacteria. The fluorescence intensity was proportional to the number of bacteria ingested. Adherent bacteria were distinguished from the ingested ones. This two‐color cellular staining permits measurement of two different functions of neutrophils in one step. This method could be of interest for the determination of the interactions between neutrophils and bacteria and for the investigations on infectious diseases in chronic granulomatous disease patients.
New chamber for flow cytometric analysis over an extended range of stream velocity and application to cell adhesion measurements
Authors: Amblard François , Cantin Claude , Durand Jocelyn , Fischer Alain , Sékaly Rafick , Auffray Charles 
Published: August 2005

AbstractWhen analyzed in a flow cytometer, particles are suddenly accelerated to high velocities (1–10 m.s−1) over very short distances. This feature is essential to obtain high analysis rates and low coincidence levels, but translates into very strong velocity gradients (> 105 s−1): particles experience strong hydrodynamic stresses that elongate them acid tend to dissociate weakly associated complexes.In order to analyze fragile conjugates formed by heterotypic adhesion between two cell types, a flow cytometer was modified to make hydrodynamic stress not only much weaker but also adjustable. A new and easily adaptable flow cell was designed for the instruments of the FACS™ series; it provided satisfactory hydrodynamic conditions on a wide continous range of flow rates. Accompanying electronic adaptations permitted standard analysis between 0.01 and 10 m.s−1.At 0.01 m.s−1, the velocity gradient roughly amounts to 50 s−1. Conjugates formed by the adhesion between human B and resting T lymphocytes, disrupted in conventional flow cytometers, could be detected and precisely quantified provided analysis velocity was kept below 0.1 m.s−1. We conclude that low velocity flow cytometry makes possible the quantification of weak intercellular adhesion phenomena, and is potentially useful for the future development of new biomechamical techniques and other applications.
Gene transfer by electroporation, lipofection, and DEAE‐dextran transfection: Compatibility with cell‐sorting by flow cytometry,
Authors: Puchalski Ralph B. , Fahl William E. 
Published: August 2005

AbstractThe aim of this work was to define a transfection procedure that is compatible with the sorting and propagation of cells that transiently express a heterologous gene. Three requirements were established for the procedure and were meet with COS monkey kidney cells that express a recombinant glutathione Stransferase (GST) gene. The transfection procedure used had to generate (i) populations in which at least 10% of the cells expressed recombinant GST, (ii) cellular morphological homogeneity throughout the population, and (iii) viable cells with at least a 5% colony‐forming ability. Of the transfection techniques tested, only electroporation satisfied all three requirements. Usually 20–22% of the cells that survived electroporation expressed recombinant GST 3 days after electroporation as measured by flow cytometry, and 25% of the cells that survived electroporation formed colonies in cloning assays. Transfection. with DEAE‐dextran and chloroquine did enable 40% of the surviving cells to express GST, but only 0.01% of the cells that survived transfection formed colonies in cloning assays. Finally, with lipofection, only 1% of the surviving cells expressed recombinant GST, although 25–40% of the cells that survived transfection formed colonies. These studies define the merits and limitations of transfection techniques relative to the analysis and sorting of transfected cells by flow cytometry.
Flow cytometric analysis of whole blood lysis, three anticoagulants, and five cell preparations
Authors: Carter Patricia H. , Resto‐Ruiz Sandra , Washington Glennelle C. , Ethridge Steve , Palini Alessio , Vogt Robert , Waxdal Myron , Fleisher Thomas , Noguchi Philip D. , Marti Gerald. E. 
Published: August 2005

AbstractWe studied the effects of anticoagulants and cell preparation methods on lymphocyte forward‐angle s catter (FSC), autofluorescence, and immunofluorescent staining for CD45, CD14, and CD13. Blood samples collected in ethylenediaminetetracetic acid (EDTA), heparin, and acid citrate dextrose (ACD) were processed by using conventional Hypaque‐Ficoll (HF) separation and four whole blood (WR) lysis techniques: Immuno‐lyse, Q‐Prep, FACS Lyse, and Gen Trak Lysis. Lymphocytes prepared by using three of the four whole blood methods gave FCS values comparable to those isolated by HF, while one method (FACS Lyse) gave consistently lover values. Autofluorescence values were comparable by all methods except Immuno‐lyse, which showed consistently higher values in blood stored for 24 h with any anticoagulant. Immunofluorescent values for CD45‐stained cells were quite consistent across all methods, and among the whole blood methods, FACS Lyse and Q‐Prep uniformly gave the highest purity of CD45‐positive cells in the lymphocyte light scatter gates. Additionally, propidium iodide (PI) analyses of CD45‐stained whole blood, and analyzed without lysis, confirmed that ACD and heparin were superior to EDTA for maintaining viable leucocytes overnight. Future studies should focus on other commonly used reagents, a wide variety of abnormal samples, and cell viability.
Rapid multivariate analysis and display of cross‐reacting antibodies on human leukocytes
Authors: Robinson J. Paul , Ragheb Kathy , Lawler Gretchen , Kelley Steve , Durack Gary 
Published: August 2005

AbstractWe present an application which can rapidly determine the binding patterns of monoclonal antibodies on mixed populations of cells simultaneously in a single rapid analysis. It is an application of the tube identifier parameter (TIP) system which can provide fully correlated listmode data of the entire patient phenotype in a single file. Using the phenogram analytical display, we are able to determine the cross‐reacting antibodies for an entire antibody panel for each cell type. This information can be displayed in a single plot. Using light scatter gating to select different populations of lymphocytes, monocytes, and neutrophils, phenograms can be simultaneously generated. This provides a directly comparable means of displaying the positive and negative binding characteristics of each antibody on each cell population. Any marker combination that is abnormal will be identifiable in the phenogram. Additionally, by plotting the fluorescence distributions of each marker beside one another (termed overview), quantifiable differences in intensity can be determined. There are 3 major benefits of the proposed analysis. By using the TIP concept, several sets of antibodies can be compared simultaneously. Any light scatter gate can be used and this gate can be changed on one histogram or plot, yet apply to the total analysis. Data analysis is particularly rapid since the entire phenotype of a patient can be evaluated by performing a single rapid analysis.
Detection of granulocyte reactive oxygen species formation in whole blood using flow cytometry
Authors: Himmelfarb Jonathan , Hakim Raymond M. , Holbrook Diane G. , Leeber Donald A. , Ault Kenneth A. 
Published: August 2005

AbstractWe have developed a technique for analysis of granulocyte reactive oxygen species formation in whole blood using flow cytometry and two color immunofluorescence. This technique relies upon the use of specific fluorescent dye (LDS‐751) to stain nucleated cells, eliminating erythrocytes from analysis. Using LDS751, forward angle light scatter, and 90° side scatter, a granulocyte gate, monocyte gate, and lymphocyte gate were identified.Analysis with multiple FITC conjugated monoclonal antibodies demonstrated greater than 95% purity of a flow cytometrically identified granulocyte population in whole blood without physical manipulation of the blood.Utilizing 2'7' dichlorofluorescein diacetate (DCFH‐DA), we were able to measure granulocyte intracellular reactive oxygen species production. Dose response curves were obtained for the effect of granulocyte agonists phorbol myristate acetate, FMLP, and heat fixed Staphylococcus aureus on reactive oxygen species production. The techniques described in this paper should be useful for measuring granulocyte activation in vivo with flow cytometry.
Multiparametric flow cytometric analysis of radiation‐induced micronuclei in mammalian cell cultures
Authors: Schreiber Georg A. , Beisker Wolfgang , Bauchinger Manfred , Nüsse Michael 
Published: August 2005

AbstractA new flow cytometric method is presented that quantities the frequency of radiation‐induced micronuclei in mammalian cell cultures with high precision. After preparing a suspension of main nuclei and micronuclei stained with ethidium bromide and Hoechst 33258, both types of particles are measured simultaneously in a flow cytometer using forward light scatter and three fluorescence emission intensities excited by UV, 488 nm, and by energy transfer from Hoechst 33258 to ethidium bromide. Nonspecific debris overlapping the micronucleus distribution especially in the low fluorescence intensity region was discriminated from micronuclei by calculating ratios of the different fhuorescences. The frequencies of radiation‐induced micronuclei measured with this new technique agreed well with results obtained by conventional microscopy. The lower limit of the DNA content of micronuclei identified by this technique was found to be about 0.5%–0.75% of the DNA content of G1‐phase nuclei. Dose effect curves and the time‐dependent induction of micronuclei were measured for two different mouse cell lines.
Comparative evaluation of several DNA binding dyes in the detection of apoptosis‐associated chromatin degradation by flow cytometry
Authors: Telford William G. , King Louis E. , Fraker Pamela J. 
Published: August 2005

AbstractMouse thymocytes readily undergo apoptosis‐associated DNA degradation upon exposure to glucocorticoids or ionizing radiation. It has been previously shown that flow cytometric cell cycle analysis of propidium iodide‐stained apoptotic thymocytes results in the appearance of a distinct cell cycle region (the A0 region) below the G0/G1 region. Cells in this region were shown to be undergoing apoptosis, and determination of apoptosis by flow cytometric analysis was proposed as a superior method for evaluating thymocyte apoptosis. In this study, a variety of DNA binding dyes with diverse primary binding mechanisms were evaluated for their ability to detect glucocorticoid and ionizing radiatiom‐induced apoptosis in mouse thymocytes. Apoptotic thymocytes stained with DNA binding dyes from the phenanthridinium, acridine, actinomycin, chromomycinone, anthracycline, and bisbenzimidazole groups all demonstrated clearly defined A0 regions with percentages comparable to those obtained for propidium iodide. These results indicate that the appearance of the A0 region is not dependent on a particular dye binding characteristic and may be the consequence of extensive changes in chromatin structure resulting in a significant degree of dye exclusion.
Novel derivation of total cell cycle time in malignant cells using two DNA‐specific labels
Authors: Bokhari S. A. J. , Raza A. 
Published: August 2005

AbstractCell cycle kinetic analysis in vitro has conventionally been accomplished by labeling S‐phase cells using two DNA specific labels given sequentially and separated from each other by a certain time interval. By counting the cells labeled by both versus those labeled by either one of the two label, and using the formulas described by Wimber and Quastler, approximate values for durations of S‐phase (Ts) and the total cell cycle (Tc) can be determined. More recently, instead of radioisotope labeled thymidine, two thymidine analogues have been used to label Sphase cells in vivo in a variety of human tumors based on the same principles. In the present report, new formulas are proposed for the calculation of Ts and Tc which are simpler to apply since only one type of labeled cells (those exiting Sphase as identified by containing only the first label) need to be differentiated from the remaining population for Tc calculations.
Flow cytometric DNA content of fresh tumor specimens using keratin‐antibody as second stain for two‐parameter analysis
Authors: van der Linden J. C. , Herman C. J. , Boenders J. G. C. , van de Sandt M. M. , Lindeman J. 
Published: August 2005

AbstractStudies concerning flow cytometric assessed DNA content reveal problems in interpretating DNA histograms of tumor specimens. The main problems are histograms with a broad coefficient of variation in the G0/G1 fraction; a high G2M fraction and samples with a low percentage of tumor cells. Therefore, in the present study, 382 fresh tumor specimens of carcinomas were analysed routinely, double labeled with, on the one hand, propidium‐iodide for assessing DNA content and, on the other, a monoclonal keratinantibody for marking epithelial and tumor cells.Of the 311 tumor samples, using single parameter analysis 165 (54%) were classified as DNA aneuploid arid 146 (46%) as DNA “euploid.” By double parameter analysis, 224 (72%) samples were keratin positive and 87 (27%) keratin negative and, of the 224 keratin positive tumors, 175 (78%) were DNA aneuploid and 49 (220) DNA euploid.The DNA histograms of single and double parameter analysis were compared and it was concluded that in 24 cases (11%) keratin labeling was necessary to recognize DNA aneuploidy. In another 23 (10%) cases, keratin labeling was helpful in assessing DNA aneuploidy. Finally when the results of the 311 samples were combined, 215 (68%) were scored as DNA aneuploid and 99 (32%) DNA euploid. Thus the overall gain in assessing DNA aneuploidy using the double labeling technique is 14%.In conclusion, it is shown that keratin labeling on fresh tumor cell suspensions of epithelial tumors is of additional value in establishing DNA content. Because single parameter DNA assessment is adequate in approximately 60% of the tested samples, the double labeling technique can be performed routinely, or after initial single parameter DNA assessment. Histograms having a broad CV and/or a high G2M are good candidates for the double labeling technique. Using this technique, DNA‐content assessment becomes more reliable.
Analysis of distribution of tumor‐ and preneoplasia‐infiltrating lymphocytes using simultaneous hoechst 33342 labeling and immunophenotyping
Authors: Loeffler David A. , Kukuruga Mark A. , Juneau Paul L. , Heppner Gloria H. 
Published: August 2005

AbstractHoechst 33342 in vivo staining was combined with immunofluorescent staining of cell surface antigens to quantify the distribution, relative to blood supply, of lymphocytes in a preneoplastic mammary lesion, the murine C4 hyperplastic alveolar nodule (HAN), and the C4 adenocarcinoma which develops from C4 HAN. The vascular supply to lymphocytes expressing Thy 1. 2, L3T4, Ly2, and ASGM1 cell surface antigens was evaluated in both tissues. The distribution of ASGM1 + cells, which include natural killer cells, differed between the two tissues, being significantly increased in the 20% brightest Hoechst‐stained lymphocyte fraction in HAN but not in C4 tumor. Distribution of T lymphocytes did not differed between the two tissues. The combination of in vivo Hoechst 33342 with in vitro immunofluorescence provides a simple method to evaluate the distribution with regard to blood supply of lymphocyte subsets in solid tumors and preneoplastic lesions.
Flow cytometric two‐color staining technique for simultaneous determination of human erythrocyte membrane antigen and intracellular malarial DNA
Authors: Pattanapanyasat Kovit , Webster H. Kyle , Udomsangpetch Rachanee , Wanachiwanawin Wanchai , Yongvanitchit Kosol 
Published: August 2005

AbstractA novel fixative and permeabilization method is described which allows simultaneous flow cytometric detection of red blood cell membrane antigen and intracellular malaria parasites. To illustrate the method, red blood cells from patients with paroxysmal nocturnal bemoglobinuria were infected with Plasmodium falciparum and maintained in synchronous red blood cell culture. The infected red blood cells were immunolabeled with antibodies directed to the complement regulatory protein decay‐accelerating factor (DAF) followed by subsequent fixations in paraformaldehyde and then glutaraldehyde in phosphate‐buffered saline. Finally, DNA of the intraerythrocytic parasites was stained with propidium iodide. Using this technique, cellular morphology was well preserved, no cell aggregation was observed, and high‐quality indirect immunofluorescence and parasite DNA staining were obtained with negligible nonspecific labelling. Simultaneous measurement of parasite DNA and red blood cell membrane determinants makes possible the investigation of alterations of red cell membrane proteins in association with development of intracellular malaria parasites.
Comparison of flow cytometry and epifluorescence microscopy for counting bacteria in aquatic ecosystems
Authors: Monfort Patrick , Baleux Bernard 
Published: August 2005

AbstractFlow cytometry was used to count bacterial cells from diverse origins: one strain of E. coli, one sample of lake water, and 18 samples of estuary water. To verify the accuracy and the precision of this technique, total bacteria counts made by flow cytometry were compared with counts by direct observation using epifluorescence microscopy. The results of this study showed that flow cytometry was a reliable technique for counting a mixture of bacteria in samples from aquatic ecosystems.