Dyes providing increased sensitivity in flow-cytometric dye-efflux assays for multidrug resistance.
AuthorsFrey T, Yue S, Haugland RP
JournalCytometry
PubMed ID7587707
In an effort to improve detection of P-glycoprotein-mediated multidrug resistance (mdr), several dyes were compared to rhodamine 123 (R123) in efflux assays. Two dyes (SY-38 and SY-3150) were found that provided better sensitivity. These dyes were effluxed by a cell line known to be mdr-positive (P388/R84) but not by an ... More
Modulation of platelet-neutrophil interaction with pharmacological inhibition of fibrinogen binding to platelet GPIIb/IIIa receptor.
AuthorsXiao Z, Théroux P, Frojmovic M
JournalThromb Haemost
PubMed ID10064007
'The study investigated how drug inhibition of the GPIIb/IIIa receptor influences the interactions between platelets and leukocytes. These interactions are believed to play an important role in the etiology of the acute coronary syndromes. Thirty patients with unstable angina or non-Q-wave myocardial infarction were studied before the administration of tirofiban ... More
Discriminating between damaged and intact cells in fixed flow cytometric samples.
AuthorsTerstappen LW, Shah VO, Conrad MP, Recktenwald D, Loken MR
JournalCytometry
PubMed ID2460298
'A vital, nucleic acid stain (LDS-751) was used to discriminate intact from damaged cells in a flow cytometer even after the samples had been fixed with paraformaldehyde. Three major cell populations with different fluorescence properties with LDS-751 were found in the fixed samples. Cells not staining or only dimly staining ... More
The Q-Prep system: effects on the apparent expression of leucocyte cell surface antigens.
AuthorsMacey MG, McCarthy DA, Davies C, Newland AC
JournalCytometry
PubMed ID9149913
'To facilitate the analysis of immunolabelled peripheral blood or bone marrow leucocytes by flow cytometry, a number of reagents are available commercially that lyse erythrocytes and fix leucocytes. This study has investigated the effect on antibody-labelled whole blood of the Q-Prep procedure, in which erythrocytes are lysed with formic acid, ... More
Interaction of LDS-751 and rhodamine 123 with P-glycoprotein: evidence for simultaneous binding of both drugs.
AuthorsLugo MR, Sharom FJ
JournalBiochemistry
PubMed ID16229491
'The P-glycoprotein efflux pump, an ABC superfamily member, can export a wide variety of hydrophobic drugs, natural products, and peptides from cells, powered by the energy of ATP hydrolysis. Transport substrates appear to first partition into the membrane and then interact with the protein within the cytoplasmic leaflet. Two drug ... More
Nucleic acid dyes for detection of apoptosis in live cells.
AuthorsFrey T
JournalCytometry
PubMed ID8582249
'Apoptotic thymocytes were found to be much dimmer than normal thymocytes when stained with several nucleic acid dyes. These dyes provide a quick and simple assay for apoptosis which works for live cells and does not require a UV laser. The collection of dyes giving this staining pattern includes reagents ... More
Isolating fetal nucleated red blood cells from maternal blood: the Baylor experience--1995.
AuthorsSimpson JL, Lewis DE, Bischoff FZ, Elias S
JournalPrenat Diagn
PubMed ID8587858
'In our previous work we have isolated fetal cells from maternal blood and used fluorescent in situ hybridization (FISH) for chromosome-specific probes to detect aneuploidy. Current efforts in the Baylor College of Medicine programme are focusing on obtaining consistency in flow-sorting methodology and on determining sensitivity and specificity. To this ... More
Neutrophil aggregation is beta 2-integrin- and L-selectin-dependent in blood and isolated cells.
AuthorsSimon SI, Chambers JD, Butcher E, Sklar LA
JournalJ Immunol
PubMed ID1383326
'Neutrophil aggregation in response to formyl peptide was analyzed in blood and isolated cells by fluorescence flow cytometry. The isolated leukocyte aggregates and the leukocytes in blood were identified with the vital nucleic acid stain LDS-751. This method enabled us to discriminate nucleated cells from other blood cells and to ... More
How should CD34+ cells be analysed? A study of three classes of antibody and five leucocyte preparation procedures.
AuthorsMacey MG, McCarthy DA, van Agthoven A, Newland AC
JournalJ Immunol Methods
PubMed ID9212835
'For patients undergoing stem cell transplantation after intensive marrow ablative therapy it is important to enumerate the CD34+ stem cells in peripheral blood so that the harvest can be timed in order to maximize the number of cells collected by leucophoresis for subsequent haematopoietic reconstitution. The use of rapid flow ... More
Bone marrow cell differential counts obtained by multidimensional flow cytometry.
AuthorsTerstappen LW, Levin J
JournalBlood Cells
PubMed ID1450429
'Five-dimensional flow cytometric analysis of normal bone marrow aspirates was utilized to determine the frequency of neutrophils, eosinophils, monocytes, lymphocytes, nucleated erythrocytes, reticulocytes, platelets, and a cell population that included blasts of each of the cell lineages, megakaryocytes, plasma cells, and basophils. Each of these bone marrow cell populations had ... More
Immunophenotyping of lymphocytes obtained by bronchoalveolar lavage: description of an all-purpose tricolor flow cytometric application.
AuthorsBrandt B, Thomas M, von Eiff M, Assmann G
JournalJ Immunol Methods
PubMed ID8690945
'A tricolor flow cytometric application is described which permits the determination of total T lymphocytes, T helper lymphocytes and cytotoxic T lymphocytes, natural killer cells and activated T lymphocytes under the same experimental conditions. Even when the lymphocyte count is low, when there is contamination by dust particles or when ... More
A rapid sample preparation technique for flow cytometric analysis of immunofluorescence allowing absolute enumeration of cell subpopulations.
AuthorsTerstappen LW, Meiners H, Loken MR
JournalJ Immunol Methods
PubMed ID2477460
'A simple and rapid method was developed for immunofluorescence measurements of cells by flow cytometry which does not require washing procedures, permitting absolute enumeration of cell subpopulations. Peripheral blood cells were labeled with fluorescein and phycoerythrin conjugated monoclonal antibodies and the nucleic acid stain LDS-751. Distilled water was added following ... More
Apoptosis of cells in aged samples as detected by the ProCOUNT reagent.
AuthorsManion K, Frey T
JournalCytometry
PubMed ID8979033
'An unusual population of high side scatter, low nucleic acid dye binding, dim CD45 cells was found in aged blood samples stained with the ProCOUNT reagent. Cell surface staining showed that these cells have the surface phenotype of neutrophils. However, they have decreased expression of several surface antigens, bind annexin ... More
Rare event selection of fetal nucleated erythrocytes in maternal blood by flow cytometry.
AuthorsLewis DE, Schober W, Murrell S, Nguyen D, Scott J, Boinoff J, Simpson JL, Bischoff FZ, Elias S
JournalCytometry
PubMed ID8974867
'A noninvasive method of prenatal genetic diagnosis requires fetal cell selection from the maternal circulation that allows efficient recovery for analysis by fluorescence in situ hybridization (FISH). We have solved several problems that negatively affect the isolation and FISH analysis of fetal nucleated red blood cells (nRBCs) in the maternal ... More
Complete remission in acute myeloid leukaemia with t(8;21) following treatment with G-CSF: flow cytometric analysis of in vivo and in vitro effects on cell maturation.
AuthorsFerrara F, Di Noto R, Viola A, Russo C, Boccuni P, Costantini S, Dello Russo A, Lo Pardo C, Del Vecchio L
JournalBr J Haematol
PubMed ID10460615
'A 75-year-old patient diagnosed as having acute myeloid leukaemia with t(8;21) received G-CSF alone as induction therapy. Complete remission was achieved following 2 weeks of treatment. Flow cytometric analysis, performed by CD45 technique modified by the introduction of preliminary gating with LDS-751, confirmed the disappearance of blast cells along with ... More
Flow cytometric analysis and modeling of cell-cell adhesive interactions: the neutrophil as a model.
AuthorsSimon SI, Chambers JD, Sklar LA
JournalJ Cell Biol
PubMed ID2277085
'The immune function of granulocytes, monocytes, lymphocytes, and other specialized cells depends upon intercellular adhesion. In many cases the molecules mediating leukocyte cell adhesion belong to the Leu-CAM superfamily of adhesive molecules. To elucidate the events of homotypic aggregation in a quantitative fashion, we have examined the aggregation of neutrophils ... More
Detection of bromodeoxyuridine incorporation by alteration of the fluorescence emission from nucleic acid binding dyes using only an argon ion laser.
AuthorsFrey T
JournalCytometry
PubMed ID7875038
'A method was developed that uses paired DNA dyes to detect the incorporation of bromodeoxyuridine (BrdUrd) into cellular DNA and requires only 488 nm excitation. The fluorescence of thiazole blue, TO-PRO-3, and LDS-751 was found to be enhanced by the presence of BrdUrd in DNA. Pairing LDS-751, thiazole blue, or ... More
Interaction of LDS-751 with P-glycoprotein and mapping of the location of the R drug binding site.
AuthorsLugo MR, Sharom FJ
JournalBiochemistry
PubMed ID15641790
'One cause of multidrug resistance is the overexpression of P-glycoprotein, a 170 kDa plasma membrane ABC transporter, which functions as an ATP-driven efflux pump with broad specificity for hydrophobic drugs, peptides, and natural products. The protein appears to interact with its substrates within the membrane environment. Previous reports suggested the ... More
Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission.
AuthorsKlar TA, Jakobs S, Dyba M, Egner A, Hell SW
JournalProc Natl Acad Sci U S A
PubMed ID10899992
'The diffraction barrier responsible for a finite focal spot size and limited resolution in far-field fluorescence microscopy has been fundamentally broken. This is accomplished by quenching excited organic molecules at the rim of the focal spot through stimulated emission. Along the optic axis, the spot size was reduced by up ... More
Flow cytometry counting of CD34+ cells in whole blood.
AuthorsFornas O, Garcia J, Petriz J
JournalNat Med
PubMed ID10888936
Flow cytometric assays of oxidative burst activity in phagocytes.
AuthorsRothe G, Valet G
JournalMethods Enzymol
PubMed ID8015489
Assessment of fluorochromes for cellular structure and function studies by flow cytometry.
AuthorsPetit JM, Denis-Gay M, Ratinaud MH
JournalBiol Cell
PubMed ID7693118
Because flow cytometry permits the analysis of individual whole cells, one of the key requirements in selecting a probe is its ability to target the site of interest into cells. In addition, dyes must possess ideal properties (ie extinction coefficient, Stoke's shift) rendering them appropriate for this methodology. Other characteristics, ... More
AuthorsKapoor V, Hakim FT, Rehman N, Gress RE, Telford WG,
JournalJ Immunol Methods
PubMed ID19268672
Telomere length analysis has been greatly simplified by the quantitative flow cytometry technique FISH-flow. In this method, a fluorescein-labeled synthetic oligonucleotide complementary to the telomere terminal repeat sequence is hybridized to the telomere sequence and the resulting fluorescence measured by flow cytometry. This technique has supplanted the traditional laborious Southern ... More
Flow cytometry and FISH to measure the average length of telomeres (flow FISH).
AuthorsBaerlocher GM, Vulto I, de Jong G, Lansdorp PM
JournalNat Protoc
PubMed ID17406480
Telomeres have emerged as crucial cellular elements in aging and various diseases including cancer. To measure the average length of telomere repeats in cells, we describe our protocols that use fluorescent in situ hybridization (FISH) with labeled peptide nucleic acid (PNA) probes specific for telomere repeats in combination with fluorescence ... More
Detection of granulocyte reactive oxygen species formation in whole blood using flow cytometry.
AuthorsHimmelfarb J, Hakim RM, Holbrook DG, Leeber DA, Ault KA
JournalCytometry
PubMed ID1372205
We 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 LDS-751, forward angle light scatter, and 90 ... More
Staining of cellular mitochondria with LDS-751.
AuthorsSnyder DS, Small PL
JournalJ Immunol Methods
PubMed ID11687236
We have found the dye LDS-751 to bind almost exclusively to mitochondria when incubated with viable, nucleated cells. Treatment of cells with the nuclear stain acridine orange and LDS-751 revealed little colocalization when the cells were examined by confocal microscopy. Staining with the dye rhodamine 123, which is known to ... More
A simple flow cytometric procedure for the determination of surface antigens on unfixed leucocytes in whole blood.
AuthorsMcCarthy DA, Macey MG
JournalJ Immunol Methods
PubMed ID7689085
A novel procedure has been developed for the quantitation by flow cytometry of function-associated antigens on neutrophils and monocytes in unlysed, unfixed, peripheral blood samples. Freshly drawn blood anticoagulated with the serine esterase inhibitor, phenylmethylsulphonyl fluoride, is mixed with the vital nucleic acid stain, LDS-751, labelled with monoclonal antibodies for ... More
Flow cytometric determination of CD11b upregulation in vivo.
AuthorsRepo H, Jansson SE, Leirisalo-Repo M
JournalJ Immunol Methods
PubMed ID8370926
We describe a flow cytometric method to evaluate upregulation of peripheral blood neutrophil and monocyte integrin CD11b in vivo. To avoid spontaneous upregulation in vitro, buffy coat cells were separated on ice and all subsequent cell handling steps were carried out at 0-4 degrees C. Such leukocytes were 95-100% viable, ... More
Beta 2-integrin and L-selectin are obligatory receptors in neutrophil aggregation.
AuthorsSimon SI, Rochon YP, Lynam EB, Smith CW, Anderson DC, Sklar LA
JournalBlood
PubMed ID7688987
We have recently found that antibodies to L-selectin, the homing receptor on neutrophils, are as effective as those to beta 2-integrin at blocking formyl peptide-stimulated aggregation. Therefore, we investigated the requirements for expression of L-selectin and beta 2-integrin on adjacent cells during aggregation. Fluorescence flow cytometry allowed characterization of aggregates ... More
Simultaneous determination of apoptosis and surface antigen expression in tumor adherent cells.
AuthorsBarzanti F, Zoli W, Susino MD, Ricotti L, Tesei A, Papa S, Renò F, Amadori D
JournalJ Biol Regul Homeost Agents
PubMed ID11860224
Apoptosis is a physiological, gene-directed form of cell death aimed at controlling cell proliferation in several biological conditions. It plays a crucial role in modulating tissue growth during embryonic development, cell turnover in adult life, and it seems to be the most frequent mechanism of tumor cell deletion by chemotherapy. ... More
Anti-D initially stimulates an Fc-dependent leukocyte oxidative burst and subsequently suppresses erythrophagocytosis via interleukin-1 receptor antagonist.
AuthorsCoopamah MD, Freedman J, Semple JW
JournalBlood
PubMed ID12829590
Previous results have demonstrated that anti-D therapy in children with chronic auto-immune thrombocytopenic purpura (AITP) induced a significant increase in several pro- and anti-inflammatory plasma cytokines within 2 hours of administration. To investigate the biologic basis of these early in vivo responses, we developed a flow cytometric assay to measure ... More
Transport of LDS-751 from the cytoplasmic leaflet of the plasma membrane by the rhodamine-123-selective site of P-glycoprotein.
AuthorsShapiro AB, Ling V
JournalEur J Biochem
PubMed ID9652412
P-glycoprotein is an ATP-dependent transporter of an extremely wide variety of lipophilic compounds. We showed previously [Shapiro, A. B. & Ling, V. (1997a) Eur. J. Biochem. 250, 130-137] that P-glycoprotein contains two drug transporting sites, dubbed H (for Hoechst 33342-selective) and R (for rhodamine-123-selective), that interact with positive cooperativity. The ... More
Multiparameter analysis of human epithelial tumor cell lines by laser scanning cytometry.
AuthorsPollice AA, Smith CA, Brown K, Farkas DL, Silverman JF, Shackney SE
JournalCytometry
PubMed ID11135288
Laser scanning cytometry (LSC) is a relatively new slide-based technology developed for commercial use by CompuCyte (Cambridge, MA) for performing multiple fluorescence measurements on individual cells. Because techniques developed for performing four or more measurements on individual lymphoid cells based on light scatter as a triggering parameter for cell identification ... More
Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry.
AuthorsO'Brien MC, Bolton WE
JournalCytometry
PubMed ID7537649
Dead cells represent a significant source of interference in the flow cytometric analysis of viable cells primarily due to nonspecific uptake of probes, increased autofluorescence, and altered antigen expression and DNA content. Traditional methods of dead cell exclusion, based on light scatter or uptake of dyes such as propidium iodide ... More
Telomere length measurement by fluorescence in situ hybridization and flow cytometry: tips and pitfalls.
AuthorsBaerlocher GM, Mak J, Tien T, Lansdorp PM
JournalCytometry
PubMed ID11813198
BACKGROUND: Telomeres containing noncoding DNA repeats at the end of the chromosomes are essential for chromosomal stability and are implicated in regulating the replication and senescence of cells. The gradual loss of telomere repeats in cells has been linked to aging and tumor development and methods to measure telomere length ... More
Effect of fixation on quantification of the expression of leucocyte function-associated surface antigens.
AuthorsMcCarthy DA, Macey MG, Cahill MR, Newland AC
JournalCytometry
PubMed ID7528123
The surface expression of adhesion molecules and other function-associated antigens on peripheral blood leucocytes may be measured by flow cytometry. However, quantification of these antigens is difficult, because their expression can be rapidly and artefactually modulated if the cells are activated in vitro. Consequently, it is common, when analyzing these ... More
Correlated flow cytometric analysis of terminal events in apoptosis reveals the absence of some changes in some model systems.
AuthorsFrey T
JournalCytometry
PubMed ID9222111
Many parallel processes occur during the final stages of apoptosis. It is not clear which of these processes occur in all or most models of apoptosis and which occur only in some. In addition, the temporal relationship of these events is not always well understood. Correlated flow cytometric measurements were ... More
Biomechanics of P-selectin PSGL-1 bonds: shear threshold and integrin-independent cell adhesion.
AuthorsXiao Z, Goldsmith HL, McIntosh FA, Shankaran H, Neelamegham S
JournalBiophys J
PubMed ID16387772
Platelet-leukocyte adhesion may contribute to thrombosis and inflammation. We examined the heterotypic interaction between unactivated neutrophils and either thrombin receptor activating peptide (TRAP)-stimulated platelets or P-selectin-bearing beads (Ps-beads) in suspension. Cone-plate viscometers were used to apply controlled shear rates from 14 to 3000/s. Platelet-neutrophil and bead-neutrophil adhesion analysis was performed ... More
Analysis of variation in results of flow cytometric lymphocyte immunophenotyping in a multicenter study.
AuthorsGratama JW, Kraan J, Van den Beemd R, Hooibrink B, Van Bockstaele DR, Hooijkaas H
JournalCytometry
PubMed ID9298834
Fifty-five laboratories participated in a send-out study of four peripheral blood samples comparing a standard protocol vs. local protocols for flow cytometric lymphocyte immunophenotyping. The standard protocol included centrally provided reagents, instrument setup using triple-fluorescent microbeads and a three-color, whole-blood immunostaining technique based on fluorescein isothiocyanate and phycoerythrin-labeled monoclonal antibodies, ... More
Active transport of fluorescent P-glycoprotein substrates: evaluation as markers and interaction with inhibitors.
AuthorsWang EJ, Casciano CN, Clement RP, Johnson WW
JournalBiochem Biophys Res Commun
PubMed ID11716514
With P-glycoprotein (P-gp) continuing to have prominence among the ABC transporters for its ability to remove various xenobiotics from many cell types, accurate and robust methods for estimating the exposure of drug, carcinogen, toxicant, pesticide, and even some endobiotics to tissues and cells affected by P-gp are valuable. The inhibition ... More
Analysis of variation in results of CD34+ hematopoietic progenitor cell enumeration in a multicenter study.
AuthorsGratama JW, Kraan J, Levering W, Van Bockstaele DR, Rijkers GT, Van der Schoot CE
JournalCytometry
PubMed ID9222096
A workshop was held in The Netherlands and Belgium with the aim of investigating whether or not the use of a standard protocol vs. local protocols for flow cytometric enumeration of CD34+ hematopoietic progenitor cells would reduce interlaboratory variation. The standard protocol consisted of a three-color, whole-blood staining technique based ... More
Multidimensional flow cytometric blood cell differentiation without erythrocyte lysis.
Forward light scattering, orthogonal light scattering, and the fluorescence intensities of unlysed peripheral blood cells, labeled with CD45-phycoerythrin and the nucleic acid dyes LDS-751 and thiazole orange, were measured simultaneously, utilizing a flow cytometer. Erythrocytes, reticulocytes, platelets, neutrophils, eosinophils, basophils, monocytes, lymphocytes, nucleated erythrocytes, and immature nucleated cells occupied unique ... More