Mucin-lectin interactions assessed by flow cytometry

The O-glycosylated domains of mucins and mucin-type glycoproteins contain 50-80% of carbohydrate and possess expanded conformations. Herein, we describe a flow cytometry (FCM) method for determining the carbohydrate-binding specificities of lectins to mucin. Biotinylated mucin was immobilized on streptavidin-coated beads, and the binding specificities of the major mucin sugar chains, as determined by GC-MS and MALDI-ToF, were monitored using fluorescein-labeled lectins. The specificities of lectins toward specific biotinylated glycans were determined as controls. The advantage of flexibility, multiparametric data acquisition, speed, sensitivity, and high-throughput capability makes flow cytometry a valuable tool to study diverse interactions between glycans and proteins.     

Analysis of phytoplankton by flow cytometry

Optical properties of eight algae species were measured on a flow cytometer. Forward and perpendicular light scatter measurements provide information on the size and shape of algae cells. The intensity of chlorophyll fluorescence varies greatly among the studied algae species and can be used to distinguish them. Measurements of chlorophyll fluorescence after excitation with different wavelengths provide a fluorescence excitation spectrum for each species over the available wavelength range. These spectra reflect the different photosynthetic pigment contents of the species. Staining algae cells with the DNA stains, Hoechst 33342 and DAPI, provides two additional optical parameters to distinguish algae populations: blue nuclear fluorescence and yellow granular fluorescence. The combination of these optical measurements enables the distinction of each algae species into a small cluster in a hyperspace of parameters. The automation of phytoplankton analysis on the flow cytometer may lead to the rapid and objective assessment of water quality.     

Characterization of cytokine interactions by flow cytometry and factorial analysis

BACKGROUND: Multiple cytokines are required for the growth and development of hematopoietic cells. The effect of many cytokines depends on the activity of other signaling pathways. These interactions are quantified using factorial experimental design and analysis. METHODS: Human umbilical cord blood (HUCB) CD34+ cells were cultured in fully defined media containing various combinations of recombinant cytokines as defined by resolution IV factorial (2(7-3)(IV)) or full factorial (2(4)) design experiments. The cytokines studied were stem cell factor (SCF), interleukin (IL)-3, megakaryocyte growth and development factor (MGDF), granulocyte-colony stimulating factor (G-CSF), Flt-3 ligand, IL-6, IL-11, and erythropoietin (EPO). In vitro cell divisions were tracked by staining CD34+ cells with 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, followed by flow cytometric analysis at 4 days of culture. In separate experiments, lineage commitment and differentiation were determined at 7 days by immunophenotype. RESULTS: In addition to the main effects of single cytokines, cytokine interactions were identified. There was a negative interaction between IL-3 and MGDF that resulted in a less than additive effect of these factors on erythroid and megakaryocytic development. The effect of Flt-3 ligand and SCF factor on CD34+ cell production was also less than additive, although the response to both cytokines was greater than single cytokines. The only positive interaction that was identified was between EPO and SCF, which resulted in the synergistic production of erythroid cells. CONCLUSIONS: Factorial analysis provides a powerful methodology to study the integration of multiple signals at the cellular and molecular level.     

Analysis of virus-infected cells by flow cytometry

Flow cytometry has been used to study virus-cell interactions for many years. This article critically reviews a number of reports on the use of flow cytometry for the detection of virus-infected cells directly in clinical samples and in virus-infected cultured cells. Examples are presented of the use of flow cytometry to screen antiviral drugs against human immunodeficiency virus (HIV), human cytomegalovirus, and herpes simplex viruses (HSV) and to perform drug susceptibility testing for these viruses. The use of reporter genes such as green fluorescent protein incorporated into HIV or HSV or into cells for the detection of the presence of virus, for drug susceptibility assay, and for viral pathogenesis is also covered. Finally, studies on the use of flow cytometry for studying the effect of virus infection on apoptosis and the cell cycle are summarized. It is hoped that this article will give the reader some understanding of the great potential of this technology for studying virus cell interactions.     

Capping of cholera toxin-ganglioside GM1 complexes on mouse lymphocytes is accompanied by co-capping of alpha-actinin

We used cholera toxin, which binds exclusively and with a high affinity to the ganglioside GM1, as a probe to investigate the distribution of this glycolipid on the surface of mouse lymphocytes. When lymphocytes are incubated with cholera toxin (or its B subunit) and then sequentially with horse anti-toxin and FITC-swine anti-horse Ig at 37 degrees C, the cholera toxin-ganglioside GM1 complex is redistributed to a cap at one pole of the cell. The capping of cholera toxin-GM1 complexes is slower than the capping of surface-Ig complexes, requires two antibodies, and is inhibited at high toxin concentrations. Cholera toxin-GM1, like surface-Ig capping, is an energy-dependent process and is inhibited by sodium azide, low temperatures, or cytochalasin B, but is unaffected by demecolcine. An affinity-purified antibody against alpha-actinin was used to examine the distribution of this cytoskeletal component during the capping process. 88% of the cells that had a surface Ig cap displayed a co-cap of alpha-actinin, and 57% of the cells that had a cholera toxin-GM1 cap displayed a co-cap of alpha- actinin. Time course studies revealed similar kinetics of external ligand cap formation and the formation of alpha-actinin co-caps. We conclude that capping of a cell-surface glycolipid is associated with a reorganization of the underlying cytoskeleton. The implications of such an association are discussed in the context of current models of the mechanism of capping.     

Analysis of malaria parasite-infected blood by flow cytometry

The use of flow cytometry in the quantitative analysis of blood from mice infected with Plasmodium vinckei has been studied. Several fluorescent dyes responsive to cell membrane potential were screened and one dye, 3,3'-dimethyloxacarbocyanine (DiOC1(3) ), was chosen for further study. Mature red blood cells (mRBC), immature RBC (imRBC), and parasitized RBC (pRBC) could be recognized and counted in the flow cytometer. When infected blood was separated on a Percoll gradient and fractions analyzed by flow cytometry using DiOC1(3), distinct populations of pRBC were recognized, the frequency of which varied with density. These subpopulations could not be correlated with distinct morphologic stages but varied with the size or age of the growing parasite. Methods combining the use of DiOC1(3) with a DNA specific-dye, Hoechst 33342, are discussed as an approach to more complete analysis of the blood of malaria-infected animals.     

The measurement of specific cell: cell interactions by dual-parameter flow cytometry

The Fc receptor-mediated aggregation of antibody-coated spleen cells with cells from the P388D1 mouse macrophage line was followed using a novel flow cytometric technique. P388D1 and spleen cells were directly labeled with green-emitting (fluorescein isothiocyanate) and red-emitting (substituted rhodamine isothiocyanate) fluorophores, respectively. They were mixed, incubated in suspension at 4 degrees C, and analyzed for aggregation with a dual laser flow cytometer. Unconjugated cells appeared as particles which were either red or green, while conjugates were detected as particles which were both red and green. Using this assay procedure, 5 X 10(4) cells were analyzed in 2-3 min for the percentages of conjugates, free spleen cells, and free P388D1 cells. Intercellular aggregation required both antibody on the spleen cells and free Fc receptors on the P388D1 cells; nonspecific aggregates accounted for 1% or less of the total particles analyzed. Measurements of the fluorescence distributions within conjugates indicated that the majority of conjugates contained a single P388D1 cell bound to 1-3 spleen cells, and that only heterophilic aggregation occurred. The flow cytometric technique described here should be applicable for the measurement of the initial events of intercellular aggregation in other systems as well.     

A microbead-based system for identifying and characterizing RNA-protein interactions by flow cytometry

We present a high throughput, versatile approach to identify RNA-protein interactions and to determine nucleotides important for specific protein binding. In this approach, oligonucleotides are coupled to microbeads and hybridized to RNA-protein complexes. The presence or absence of RNA and/or protein fluorescence indicates the formation of an oligo-RNA-protein complex on each bead. The observed fluorescence is specific for both the hybridization and the RNA-protein interaction. We find that the method can discriminate noncomplementary and mismatch sequences. The observed fluorescence reflects the affinity and specificity of the RNA-protein interaction. In addition, the fluorescence patterns footprint the protein recognition site to determine nucleotides important for protein binding. The system was developed with the human protein U1A binding to RNAs derived from U1 snRNA but can also detect RNA-protein interactions in total RNA backgrounds. We propose that this strategy, in combination with emerging coded bead systems, can identify RNAs and RNA sequences important for interacting with RNA-binding proteins on genomic scales.     

Analysis of ploidy in a planarian by flow cytometry

Flow cytometry (flow microfluorimetry) provides a quick means for analysis of ploidy in planarians. Nuclei from homogenized tissues of the freshwater planarian Dugesia japonica japonica Ichikawa et Kawakatsu were stained with propidium iodide and measured with an argon-laser flow cytometer to produce histograms of DNA content. Tissues from sexually mature individuals produced histograms with a 1n (haploid) peak but no 3n peak (triploid peak), whereas those from asexual individuals showed a 2n peak or a 3n peak or both, but no 1n peak. Thus, the 1n peak distinguished sexual individuals. Mixoploid individuals, i.e., mosaics with both diploid and triploid tissues, were identified by the presence of both a 2n peak and a 3n peak. The ratios of the heights of the 2n and 3n peaks from tissues in different parts of a single mixoploid individual were similar, suggesting that the diploid and triploid cells are homogeneously distributed.     

Analysis of repetitive DNA in chromosomes by flow cytometry

We developed a flow cytometry method, chromosome flow fluorescence in situ hybridization (FISH), called CFF, to analyze repetitive DNA in chromosomes using FISH with directly labeled peptide nucleic acid (PNA) probes. We used CFF to measure the abundance of interstitial telomeric sequences in Chinese hamster chromosomes and major satellite sequences in mouse chromosomes. Using CFF we also identified parental homologs of human chromosome 18 with different amounts of repetitive DNA.     

Analysis of high-pressure-induced disruption of human erythrocytes by flow cytometry

High-pressure-induced hemolysis is suppressed by pretreating human erythrocytes at 49 degrees C, or enhanced by pretreatment with trypsin. So, the response of these pretreated cells to a pressure of 200 MPa was examined using flow cytometry. In the case of intact erythrocytes, a major product was fragmented particles. From 49 degrees C-pretreated cells, vesicles were mainly released. Trypsin-pretreated cells mainly produced open ghosts. Additionally, intact erythrocytes, 49 degrees C-pretreated ones, and trypsin-pretreated ones also released at 200 MPa vesicles of diameter 464 +/- 9, 259 +/- 18, and 574 +/- 16 nm, respectively. These results suggest that mother cells, fragmented particles, vesicles, and open ghosts from 200 MPa-treated erythrocytes are easily monitored by flow cytometry and that the size of released vesicles may also be an important factor in high-pressure-induced hemolysis.     

Analysis of coccidian oocyst populations by means of flow cytometry

Flow cytometry was employed as a tool to analyze and characterize batches of oocysts from laboratory and field isolates of Eimeria spp. from chickens and to propagate sub-populations of batches of oocysts. Oocyst batches were cleaned of debris by a combination of salt flotation, washing and treatment with dilute sodium hypochlorite (1.5% aqueous). Oocyst size and shape were registered by forward-angle light scatter with the argon laser excitation set at 488 nm at 300 mW. Sub-populations of oocysts were collected by map gating and used for microscopy or for propagation. The profile of particle size was characteristic for each species. Propagation of sub-populations of oocysts of specified sizes resulted in cultures of coccidia that were pure species or nearly pure species. The small size of E. mitis caused difficulty in separation from the remaining fine debris. This technique was useful for studying the mixed isolates by bit-map gating had the same limitations as micromanipulation because of the overlapping size of Eimeria spp. Characterization is further limited by the lack of suitable size/shape standards for flow cytometry.     

Analysis of the chromosome number in cells by flow cytometry

A new flow-cytometric method is described that permits analysis of the number of chromosomes of individual cells. Preliminary stained mitotic cells are passed through a specially designed flow chamber in which they are destroyed just before passing through the focused laser beam of the flow cytometer. Signals from chromosomes of the destroyed cells are counted, and the results are represented as a distribution of the number of chromosomes in the cells. The method may be applied for the detection of relatively rare cells with abnormal numbers of chromosomes in biological and clinical research.     

Analysis of MOPC-315 plasmacytoma by elutriation and flow cytometry

Intravenously transplanted murine plasmacytoma MOPC-315 cells were separated from normal spleen cells from a tumour-bearing mouse by elutriation and characterized according to morphology, immunologic properties and clonogenicity. Morphologically, both lymphocytoid and plasmacytoid cells were separable by elutriation. Flow cytometry correlated DNA content and intracytoplasmic IgA content and demonstrated two distinct populations, both in cell cycle, but with markedly different cellular IgA levels. Density gradient separation characterized the lower-density cells with lower IgA content and higher clonogenicity. From these studies a model of cellular differentiation is proposed.     

Analysis of apoptosis by propidium iodide staining and flow cytometry

Since its introduction, the propidium iodide (PI) flow cytometric assay has been widely used for the evaluation of apoptosis in different experimental models. It is based on the principle that apoptotic cells, among other typical features, are characterized by DNA fragmentation and, consequently, loss of nuclear DNA content. Use of a fluorochrome, such as PI, that is capable of binding and labeling DNA makes it possible to obtain a rapid (the protocol can be completed in about 2 h) and precise evaluation of cellular DNA content by flow cytometric analysis, and subsequent identification of hypodiploid cells. The original protocol enhanced the capacity for a rapid, quantitative measure of cell apoptosis. For this reason, since its publication, the PI assay has been widely used, as demonstrated by the large number of citations of the original paper and/or the continuous use of the method in many laboratories.     

Analysis of chromosomes from human peripheral lymphocytes by flow cytometry

A method of preparation and flow cytometric analysis of chromosomes from human peripheral lymphocytes is described. The procedure allows a resolution coefficient of variation better than 3% using propidium iodide staining and a commercially available flow cytometer.     

Phytoplankton monitoring by flow cytometry

The application of flow cytometry to the monitoring of phytoplanktonis demonstrated. A comparison is made with conventional approachesto phytoplankton monitoring: light microscopy for the determinationof species abundance, and chlorophyll a determination and insitu chlorophyll a measurement by fluorescence for the determinationof the biomass. Flow cytometric measurements correlate wellwith these conventional types of measurements, as has been shownby comparing a full year of monitoring data obtained at a fixedmonitoring location 10 km off the Dutch coast. Flow cytometrybridges the gap between labour-intensive, but highly informative,microscopic observations and simple biomass measurements withless information content: via flow cytometry optical data areobtained at high speed for individual particles, which can betranslated into biomass information. On the basis of the flowcytometric measurements, rough discrimination of phytoplanktonspecies groups is possible, particularly for the abundant species.Of crucial importance is careful calibration of the flow cytometer,to ensure quantitative and comparable measurements over a longperiod of time. Calibration and quality assurance aspects arecovered in detail. ³Present address: Akzo Nobel Central Research Laboratories Arnhem,Department CRL, PO Box 9300, NL-6800 SB Arnhem, The Netherlands