Impact of coincidence on granulocyte-platelet complex determination by flow cytometry is evaluated by a novel computer simulation model of coincidence

Cell complexes composed of two different cells labeled with different fluorophores can be detected as double positive events in the flow cytometer. Double positivity can originate not only from real complexes but from non-interacting coinciding cells as well. Coincidence has a high impact on the determination of the amount of platelet–granulocyte complexes since platelet concentration is in the orders of magnitude higher than that of the granulocytes. A computer model has been developed to simulate coincidence in the flow cytometer to reveal the contribution of coincidence to the overestimation of the total amount of platelet–granulocyte complexes. Mixtures of non-interacting fluorescent beads as well as EDTA-anticoagulated blood samples were analyzed in the flow cytometer.

An excellent fit was found between computer simulated and measured data pairs. Bead mixture in the flow cytometer and simulation of that resulted in 37.3 ± 1.3 and 35.7 ± 0.6% double positivity, respectively. 30.2 ± 4.3% double positivity was measured for EDTA-anticoagulated blood samples while simulation of that resulted in 28.3 ± 0.6%. Double positivity attributed to platelet–granulocyte complexes in slightly diluted blood samples might originate in coincidence and not from true complexes.     

Wide applicability of a flow cytometric assay to measure absolute membrane potentials on the millivolt scale

To prove the general applicability of a recently published flow cytometric method to determine the membrane potentials of cells on the absolute (mV) scale, the validity of the premises involved were analyzed individually. Experimental evidence was given for bis-oxonol, the applied membrane potential indicator being a Nernstian dye. The results of special measurements proved that extracellular dye concentrations were not affected by cellular dye uptake under the applied experimental conditions and that the dye content of the suspending medium did not contribute directly to the measured cellular fluorescence. A direct, membrane-potential-independent contribution of the extracellular dye to cellular fluorescence was also found to be negligible, as membrane potential values of the same type of cells evaluated from measurements in the presence of different extracellular oxonol concentrations were very close to each other. The transmembrane potential of B lymphoid JY cells was measured by the method as a function of cell density in the tissue culture. Cells isolated during the log phase of growth displayed a –40±4 mV membrane potential. At a high density of the culture (plateau phase), a significant increase of the membrane potential to –61±3 mV was observed and a medium value of –47±3.5 mV was measured at an intermediate density of the cells. Our observation indicates that nonadherent cells can also be hyperpolarized when optimal growth conditions are terminated. Received: 14 April 1998 / Revised version: 22 June 1998 / Accepted: 16 July 1998     

Measurement of the carbohydrate-binding specificity of lectins by a multiplexed bead-based flow cytometric assay

Carbohydrate binding underlies many cell recognition events. Here, we describe a multiplexed glyco-bead array method for determining the carbohydrate-binding specificities of plant lectins using a bead-based flow cytometric analysis. N-glycans including high mannose, hybrid, and complex types and O-glycans from glycoproteins were immobilized on multiplexed beads, and the specificities of 13 kinds of sugar chains were monitored within 2 h in a single reaction. This strategy is easy, rapid, reproducible, and suitable for small samples and allows the reliable and simultaneous elucidation of sugar-binding properties under identical conditions.     

Standardization of a method to detect bovine sperm-bound antisperm antibodies by flow cytometry

The objectives were to standardize some methodological and analytical aspects of a direct technique to detect sperm-bound antisperm antibodies (ASAs) in bovine semen using flow cytometry, including the effects of prefixation of sperm membranes with formalin buffer solution and inclusion of dead cells in the analysis. Fourteen Angus bulls, including ASA-positive (experimentally induced ASAs) and 10 reproductively normal ASA-negative bulls, were used. Fixation of sperm membranes had no significant effect on the percentage of IgG- or IgA-bound spermatozoa detected by flow cytometry. However, including dead cells in the analysis increased the percentage of IgG-bound spermatozoa in fixed (live and dead 18.6 ± 9.7% and live 1.3 ± 0.5%; median ± SEM) and nonfixed samples (live and dead 18.8 ± 9.2%, live 1.5 ± 0.6%; P = 0.0029), as well as IgA-bound spermatozoa in fixed (live and dead 16.3 ± 6.4%, live 0.3 ± 0.5%) and nonfixed samples (live and dead 21.4 ± 4.6%, live 1.0 ± 0.5%; P = 0.0041) in semen from ASA-negative bulls. Intrasample, intra-assay, and interassay coefficients of variation (CV) were 0.8%, 4.6%, and 5.3%, respectively, for determination of sperm-bound IgG, and were 2.8%, 8.4%, and 40.3% for determination of sperm-bound IgA. Despite the high interassay CV for IgA determination, all ASA-positive bulls consistently had high percentages of IgA-bound spermatozoa. Flow cytometry correctly identified ASA-positive bulls. Confocal laser microscopy confirmed binding of ASAs to sperm heads and cytoplasmic droplets, and less frequently to midpieces and principal piece. In conclusion, although fixation was not necessary, dead cells should be excluded from the analysis, because ejaculates with a large proportion of dead cells can yield false-positive results. Flow cytometry was accurate and reliable for detection of sperm-bound IgG and IgA and discrimination between ASA-positive and ASA-negative bulls.     

Synthesis of purine-scaffold fluorescent probes for heat shock protein 90 with use in flow cytometry and fluorescence microscopy

Fluorescent ligands for the heat shock protein 90 (Hsp90) were synthesized containing either fluorescein isothiocyanate (FITC), 4-nitrobenzo[1,2,5]oxadiazole (NBD) or the red shifted dye sulforhodamine 101 (Texas Red) conjugated to PU-H71. Two of the compounds, PU-H71-FITC2 (9) and PU-H71-NBD1 (8), were shown to be suitable for fluorescence-activated flow cytometry and fluorescence microscopy. Thus these molecules serve as useful probes for studying Hsp90 in heterogeneous live cell populations.     

Application of flow cytometry to the study of antiphagocytic properties of Klebsiella pneumoniae capsular polysaccharide

Abstract We used flow cytometry to compare the effects of whole cells and capsular polysaccharides of Klebsiella pneumoniae on the phagocytic ability ot polymorphonuclear leukocytes. Our results showed a light descrease in phagocytic activity in the presence of capsular polysaccharides, but a marked decrease with whole cells. Our findings suggest that the resistance to phagocytosis in these microorganisms is not due exclusively to their capsule, as claimed by other authors.     

A flow-cytometric method for determination of yeast viability and cell number in a brewery

A flow-cytometric assay, using the fluorescent dye, oxonol, for the simultaneous determination of yeast cell viability and cell number is described. The assay was optimised, and trialed at a brewery for 6 months. The flow-cytometry assay offered a substantially reduced error in viability determination, compared to methylene blue which is the industry standard for measuring viability. Further, by calculating yeast cell number at the same time, this assay provides a reliable method for determining pitching rate, allowing increased quality control of subsequent fermentations.     

NucleoCounter—An efficient technique for the determination of cell number and viability in animal cell culture processes

The NucleoCounter is a novel, portable cell counting device based on the principle of fluorescence microscopy. The present work establishes its use with animal cells and checks its reliability, consistency and accuracy in comparison with other cytometric techniques. The main advantages of this technique are its ability to handle a large number of samples with a high degree of precision and its simplicity and specificity in detecting viable cells quantitatively in a heterogeneous culture. The work addresses and overcomes the problems of subjectivity, and some of the inherent sampling errors associated with using the traditional haemocytometer and Trypan Blue exclusion method. NucleoCounter offers reduced intra- and inter-observer variation as well as consistency in repetitive analysis that establishes it as an efficient and highly potential device for at-line monitoring of animal cell processes. Furthermore, since the only manual steps required are sample aspiration and mixing with two reagents, it is feasible that the whole method could be automated and brought on-line for process monitoring and control.     

A quantitative method to study co-adhesion of microorganisms in a parallel plate flow chamber: basic principles of the analysis

Intermicrobial aggregation is described as one of the factors contributing to dental plaque formation. Intermicrobial aggregation is usually measured by mixing potential partners suspended in a liquid phase (‘coaggregation’). However, even if aggregation in the liquid phase occurs, adhesion of microorganisms to partners already adhering to a substratum surface may also occur (‘co-adhesion’). Coaggregation assays have been performed in order to measure coaggregation and to model co-adhesion, although it is not yet clear which the two prevails in vivo. Apart from being semi-quantitative (scores run from 0 to 4) it is questionable whether coaggregation assays really mimic co-adhesion. This study was designed to develop a method to quantitative assess co-adhesion of microbial pairs in order to gain a better understanding of the mechanisms governing co-adhesion. Co-adhesion of coaggregating and non-coaggregating partners (S. oralis, S. sanguis and A. naeslundii) to glass has been studied in a parallel plate flow chamber using real time image analysis. The spatial arrangements of adhering bacteria were analyzed by radial pair distribution functions, revealing the relative density of adhering bacteria around adhering bacteria from the same (g₂₂(r)) or a partner strain (g₂₁(r)). Pair distribution functions g₂₁(r) of coaggregating pairs clearly reveal a preference of coaggregating streptococci (S. oralis J22 and S. sanguis PK2951) to adhere around the actimomyces (A. naeslundii PK213 or T14V-J1), which were used to coat the bare glass substratum. Besides, the distribution function g₂₁(r) showed differences in co-adhesion patterns for strains with the same coaggregation score. From the results presented in this paper it can be concluded that with a parallel plate flow chamber, co-adhesion can be quantified on a continuous scale under well controlled conditions, more closely resembling those occurring in vivo.     

Development of a flow cytometric assay to study glucocorticoid receptor-mediated gene activation in living cells

A flow cytometry-based reporter gene assay was developed and utilized to measure glucocorticoid receptor (GR)-mediated gene activation at the single cell level in living cells. A reporter gene was generated that contains two copies of the glucocorticoid response element and an E1b TATA box upstream of a destabilized enhanced green fluorescent protein. Glucocorticoid activation of the reporter gene in Cos-1 and HTC cell lines was measured in vivo by flow cytometry and was shown to be dose dependent, leading to an increase in total fluorescence of the cell population. Flow cytometric analysis indicated this increase in total fluorescence per sample resulted from an increase in the number of cells expressing the activated green fluorescent protein (GFP) reporter as well as an overall increase in the mean GFP fluorescence within cells. Activation of reporter gene activity was time dependent occurring as early as 1-2h after dexamethasone addition. Activation of the reporter gene was specific as it exhibited different sensitivities to a range of glucocorticoids and activation could be blocked with glucocorticoid receptor antagonists. Coexpression of the coactivator SRC-1a or P65 subunit of NF-kappa B with GR led to enhancement or repression, respectively. Taken together, these data suggest the reporter-based flow cytometry assay is an effective method for analyzing glucocorticoid receptor-mediated gene expression at the single cell level in living cells.     

Fluorescent <Emphasis Type="Italic">in situ</Emphasis> hybridization and flow cytometry as tools to evaluate the treatments for the control of slime-forming enterobacteria in paper mills

Slime formation is a serious problem nowadays in the paper industry. Some enterobacteria are associated with the formation of slime deposits in paper and board mills. Detection and characterization of slime forming bacteria, belonging to the genus Enterobacter, Raoultella, and Klebsiella have been achieved by fluorescence in situ hybridization (FISH), using one probe based on the enterobacterial repetitive intergenic consensus sequence and other two rRNA targeted oligonucleotide probes. The effects of three kinds of antimicrobiological products (biocides, dispersants, and enzymes) on these enterobacterial cells were analyzed by flow cytometry (FC). Biocides Butrol 1009 and 1072 were the most effective microbiocides against all enterobacterial cells analyzed, reaching 90% of dead bacteria after 24 h. However, the enzymatic treatment (Buzyme) was not equally efficient on enterobacteria and its microbiocide capacity varied depending on the type of microorganism. FISH and FC were effective tools to detect important slime forming enterobacteria and to select specific treatments to control microbial problems in the paper industry.     

Evaluation of the Litron In Vitro MicroFlow Kit for the flow cytometric enumeration of micronuclei (MN) in mammalian cells

We have evaluated the performance of the prototype In Vitro MicroFlow^® Kit (Litron Laboratories), which offers a flow cytometric method for scoring micronuclei (MN). This method uses sequential staining to differentiate MN from chromatin fragments derived from apoptotic or necrotic cells. Data were generated using the genotoxins methylmethane sulphonate (MMS), dimethylbenzanthracene (DMBA) and vinblastine, and the non-genotoxins dexamethasone and staurosporine, which are known to induce apoptosis in vitro. The results obtained with these agents were compared with conventional microscopy.For short-duration exposures (3–4 h) both manual and flow methodologies demonstrated good concordance, with concentration-related increases in the percentage of MN for MMS, DMBA and vinblastine. Statistically significant increases were observed at ≥20 and 40 μg/mL, for manual and flow analysis, respectively, for MMS; at 0.5 and 0.75 μg/mL for DMBA; and at 0.035 and 0.04 μg/mL, respectively, for vinblastine. Dexamethasone showed clear negative responses by manual and flow cytometric analysis, with comparable results for both methodologies (all <1.7-fold compared with concurrent vehicle controls). Data for staurosporine, however, were less consistent showing significantly higher flow cytometric MN frequencies compared with those seen after manual analysis.Continuous (24 h) treatments were also conducted with MMS, vinblastine, dexamethasone and staurosporine. There was good concordance between the methodologies for MMS, staurosporine and vinblastine. However, dexamethasone generated discordant results, i.e. microscopic analysis was clearly negative at all doses tested, whereas flow cytometry produced significant increases in MN frequency (up to 8.1-fold at 100 μg/mL compared with the concurrent vehicle control).The inconsistencies observed between flow cytometry and standard microscopy, and the differences in assay sensitivity, particularly for apoptosis-inducing compounds, suggest that the prototype In Vitro MicroFlow^® Kit requires further refinement. Studies to investigate new parameters to address these issues are now under way and will be reported separately.     

Use of bicistronic vectors in combination with flow cytometry to screen for effective small interfering RNA target sequences

The efficacy and specificity of small interfering RNAs (siRNAs) are largely dependent on the siRNA sequence. Since only empirical strategies are currently available for predicting these parameters, simple and accurate methods for evaluating siRNAs are needed. To simplify such experiments, target genes are often tagged with reporters for easier readout. Here, we used a bicistronic vector expressing a target gene and green fluorescent protein (GFP) to create a system in which the effect of an siRNA sequence was reflected in the GFP expression level. Cells were transduced with the bicistronic vector, expression vectors for siRNA and red fluorescent protein (RFP). Flow cytometric analysis of the transduced cells revealed that siRNAs for the target gene silenced GFP from the bicistronic vector, but did not silence GFP transcribed without the target gene sequence. In addition, the mean fluorescence intensities of GFP on RFP-expressing cells correlated well with the target gene mRNA and protein levels. These results suggest that this flow cytometry-based method enables us to quantitatively evaluate the efficacy and specificity of siRNAs. Because of its simplicity and effectiveness, this method will facilitate the screening of effective siRNA target sequences, even in high-throughput applications.     

Variations in flow cytometric forward scatter signals and cell size in batch cultures of Escherichia coli

Abstract Flow cytometry was used to study the lag, exponential, stationary and death phases of non-fixed cultures of Escherichia coli . Fluctuations in the forward angle scatter signal (FALS) were compared with cell size as measured by scanning electron microscopy at low temperature and image analysis. A correlation between FALS and cell size was not observed, although a correlation (r = −0.8) was obtained between FALS and the age of the culture for the first eleven days of incubation. Marked increases in FALS were observed during the lag phase, which were attributed both to changes in size and changes in structure or chemical composition. The distribution of FALS for all culture phases was asymmetric, and was associated with the cell size distribution.     

An efficient method for flow cytometric analysis of pollen and detection of 2n nuclei in Brassica napus pollen

A simple and reliable method was developed for isolating pollen nuclei from Brassica napus and Triticum aestivum for DNA analysis using flow cytometry. The nuclei were released from pollen by ultrasonic treatment. The isolated nuclei following filtration through nylon mesh and a purification procedure were suitable for flow cytometric analysis as well as for isolating genomic DNA. Ultrasonic treatment time was optimized for B. napus pollen at different developmental stages. The method is effective and suitable for the preparation of many samples. We analyzed the nuclear DNA levels in pollen of B. napus at three major developmental stages as well as in mature wheat pollen. Only a single 1C peak representing the haploid DNA level was detected in the nuclei isolated from Brassica uninucleate microspores as well as in mature Triticum pollen. Interestingly, diploid nuclei were detected in both binucleate and mature pollen of B. napus. The possible origins of the diploid nuclei are discussed.Abbreviations DAPI 4,6-Diamidino-2-phenylindole - NIB Nuclear isolation buffer     

A statistical theory for flow cytometry profiles in terms of the binding of ligands to cell surface receptors and changes in gene expression

Flow cytometry analysis is a technique used for obtaining light scattering and fluorescence intensity data in order to characterise a chosen cell line. From a sample of the data obtained, it is desired to infer the distribution of cell size, cell granularity and occupancy of cell surface receptors, by constructing histograms for the variables of interest. Often an attempt is made, for instance, to account for the changes in shape of these histograms in terms of alterations in gene expression, etc. In this paper we analyse the way that changes in the sample histograms can be interpreted in three frequently encountered situations, namely (a) when there is one cell line exposed to alterations in chemical potential of ligand, (b) when there are two cell lines exposed separately to saturating concentrations of the same ligand, and (c) when two ligands are added in saturating amounts, first separately, then together, to the same cell line. We demonstrate that, under a wide range of assumptions, the change in histogram shape can be accounted for in terms of a proportionate and absolute component and examples are given to illustrate this. Finally, a computer program to analyse experimental data in terms of estimated shift and stretch parameters is described.     

A reverse effect of in vitro and in vivo concanavalin A-induced spleen cells on anti-sheep red blood cells response detected with a new method based on flow cytometry

A simple flow cytometric method for detecting humoral immunity against sheep red blood cells (SRBC) is described. The SRBC were incubated with the serum from SRBC-immunized mice, monoclonal anti-SRBC, or the supernatant which was obtained from the in vitro primary culture of spleen cells with SRBC. The antibodies which bound to SRBC were estimated by means of an immunofluorescence and a flow cytometry. When the channel number of the peak in the histogram of flow cytometry was measured as an index of fluorescence intensity of SRBC, the number significantly correlated with the concentration of IgM and IgG classes of anti-SRBC. The flow cytometry method and hemagglutination (HA) test, as a classic method, were compared in SRBC-immune sera and monoclonal anti-SRBC antibody. The sensitivity determined with flow cytometry was much higher than that with HA. The minimum detectable concentration of anti-SRBC antibody was found to be 3.4 ng/ml by the flow cytometry. The dose response of SRBC in in vitro primary culture was detected by the flow cytometry, not by HA, and the response increased with the dose of SRBC. Using this method, the effect of in vitro and in vivo concanavalin A (Con A)-induced spleen cells on humoral response against SRBC was examined in an in vitro culture system. Anti-SRBC response (IgM and IgG) was found to be suppressed by in vitro Con A-induced lymphocytes, but enhanced by in vivo Con A-induced lymphocytes. Thus, this new approach is found to be a good method for detecting the in vitro primary humoral antibody response, which is known to have a low reactivity.     

The changing pattern of "smart" flow cytometry (S-FC) to assist the cost-effective diagnosis of HIV, tuberculosis, and leukemias in resource-restricted conditions

There is a need to introduce cytometry into areas of the globe that have remained virtually untouched by modern laboratory medicine. With the demand to carry out tests on 100,000 s of individuals requiring antiretroviral therapy (ART), flow cytometry must remain simple and cost-effective - while being sustainable and industry supported as well as proven by quality assessment (QA). This outlook is referred to as "smart flow cytometry" (S-FC). There are five main areas where the power of S-FC is demonstrated. These are: (i) the use of CD45 to assist precise cell counting in blood and tissue samples; (ii) the primary CD4 gating to count CD4+ T cells in patients waiting for ART, including the combination (i) and (ii) in the panleucogating (PLG) protocol; (iii) monitoring of human immunodeficiency virus (HIV+) patients during ART by the decreasing levels of lymphocyte activation in a CD8/CD38 test - leading to economies of viral-load assays; (iv) in tuberculosis and HIV-TB coinfections the use of TB-antigen-stimulated cytokine-synthetic CD4+ T cells to identify active disease; and (v) the utilization of "minimal residual disease (MRD)-Lite" technology in patients 19 days after the start of antileukemic therapy to detect MRD. These methods of S-FC have been successfully introduced in "resource-restricted" countries with international and local QA.