Binding of rough lipopolysaccharides (LPS) to human leukocytes. Inhibition by anti-LPS monoclonal antibody

The binding of rough LPS (ReLPS from Salmonella minnesota R595) to human peripheral blood polymorphonuclear leukocytes (PMN), monocytes, and lymphocytes was examined by using fluorescein-labeled LPS and flow cytometry. At 4 degrees C, FITC-ReLPS bound rapidly in a concentration- and time-dependent way to PMN, monocytes, and lymphocytes. Because mononuclear cells showed both binding and nonbinding cell populations, FITC-ReLPS was used in conjunction with specific phycoerythrin-labeled mAb to identify these cell subpopulations. In contrast to T lymphocytes and NK cells, all monocytes and B lymphocytes efficiently bound FITC-ReLPS. PMN and monocytes showed two to three times more cell-associated FITC-ReLPS when cells were incubated at 37 degrees C compared with incubation at 4 degrees C. Binding of FITC-ReLPS to lymphocytes was similar for both 4 degrees C and 37 degrees C incubation conditions. In contrast to 4 degrees C, at 37 degrees C cell-associated LPS reflects surface-bound as well as internalized LPS, as demonstrated with fluorescence quenching of extracellular FITC-ReLPS by trypan blue. At 4 degrees C, binding of FITC-ReLPS was inhibited by polymyxin B. In addition, purified IgM mAb directed against hydrophobic acyl residues of ReLPS showed more than 95% inhibition of ReLPS binding to leukocytes, indicating the ability of specific mAb to prevent LPS-cell interactions necessary to exert biologic effects. The use of mAb, directed against different parts of the LPS molecule, provides an alternative method for LPS binding-inhibition studies.     

Flow-cytometric Studies of the Phagocytic Capacities of Equine Neutrophils

Methodological aspects of flow-cytometric evaluation of the phagocytic properties of equine neutrophils were elucidated. The kinetics of attachment and ingestion were studied, and the phagocytic process was more rapidly completed when serum-opsonized yeast cells were used than with use of IgG-opsonized yeast cells. Trypan blue was successfully used to quench fluorescence of non-ingested yeast cells. There were only minor differences in the kinetics of phagocytosis between quenched and un-quenched samples, indicating that attachment is rapidly followed by ingestion. Trypan blue quenching caused loss of cells with light scattering properties of granulocytes, although this did not affect the determined frequencies of truly phagocytic neutrophils. Aggregation of yeast cells proved to be a disturbance but not an obstacle to the determination of frequencies of actively phagocytic cells. Flow cytometry is well suited for studies of phagocytosis of yeast cells by equine neutrophils, and the trypan blue quenching provides a means of eliminating false-positive events due to aggregation of yeast cells. The main advantage of the flow-cytometric method is the possibility of rapid processing of a large number of samples, making the method useful for studies of herds.     

Inactivation during phagocytosis of leucine aminopeptidase, an ecto-enzyme of polymorphonuclear neutrophils

Changes in enzyme activities of the plasma membrane makers were examined during phagocytosis using guinea-pig polymorphonuclear neutrophils. Incubation of neutrophils with fresh serum-opsonized zymosan particles showed a significant reduction in leucine aminopeptidase activity, whereas 5′-nucleotidase and alkaline phosphodieterase activities remained unchanged. Inactivation of leucine aminopeptidase activity was also observed by exposure of neutrophils to complement-opsonized zymosan particles, but not to non-opsonized zymosan, IgG-coated zymosan or polysterene latex particles. Pretreatment of neutrophils with cytochalasin B, which prevents phagocytosis but not surface binding of particles, provoked inactivation to the same degree as when the cells were allowed to phagocytose the particles. However, the inactivation during phagocytosis was protected by serine protease inhibitors. These findings suggest that loss of leucine aminopeptidase activity from phagocytosing cells may be mediated by certain serine protease inhibitor-sensitive factor(s) which are probably activated by the attachment of an opsonized zymosan particle to a specific membrane receptor, probably the C3b receptor.     

Adherence of human neutrophils changes Ca signaling during activation with opsonized particles

Changes in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) upon activation of human neutrophils by opsonized particles (serum-treated zymosan; STZ) were evaluated by three different methods: (i) measurement of total fluorescence changes in indo-1 loaded neutrophils activated in suspension; (ii) measurement of fluorescence changes in individual indo-1 loaded neutrophils in a flow cytometer and (iii) measurement of fluorescence changes in individual fura-2 loaded neutrophils adherent to serum-coated coverslips. Our study shows that the opsonized particle-induced change in [Ca²⁺]_i in neutrophils is altered during adherence of the cells to a serum-coated surface. These observations might be of importance for neutrophil function in vivo, since adherence is a prerequisite for diapedesis and chemotaxis.     

Perturbation of beta-glucan receptors on human neutrophils initiates phagocytosis and leukotriene B4 production

Normal human neutrophils were stimulated with the yeast cell wall product, zymosan, and examined for two biologic responses, ingestion of particles and production of leukotriene B4 (LTB4), under conditions that were comparable and optimal for the quantitation of each response. Monolayers of adherent neutrophils ingested unopsonized zymosan particles, at particle-to-cell ratios of 12.5:1 to 125:1, in a dose- and time-related manner. At a ratio of 125:1, the percentages of neutrophils ingesting greater than or equal to 1 and greater than or equal to 3 zymosan particles reached plateau levels of 55 +/- 6 and 32 +/- 9% (mean +/- SD, n = 8), respectively, within 30 min. At this same ratio, neutrophils during gravity sedimentation with zymosan particles synthesized LTB4 in a time-dependent manner for at least 45 min. The maximum amount of immunoreactive LTB4 released into supernatants was 3.8 +/- 1.2 ng per 10(6) neutrophils (mean +/- SD, n = 5) and the corresponding total immunoreactive LTB4 was 6.2 +/- 1.9 ng per 10(6) neutrophils. Treatment of 2 x 10(7) suspended neutrophils with 250 micrograms of trypsin for 20 min before concurrent assessment of neutrophil phagocytosis and LTB4 production reduced both of these responses by about 50%. Pretreatment of neutrophils with 800 micrograms/ml of soluble yeast beta-glucan inhibited their ingestion of zymosan by 84% (mean +/- SD, n = 3), with 50% inhibition occurring with 100 micrograms/ml of soluble beta-glucan; 800 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. Pretreatment of neutrophils with 400 micrograms/ml of soluble yeast beta-glucan inhibited neutrophil synthesis of LTB4 by 90%, with 50% occurring with 200 micrograms/ml; 400 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. The presence of 1.25 micrograms/ml of cytochalasin B during incubation with zymosan particles reduced neutrophil phagocytosis from 65 to 6%, and neutrophil synthesis of LTB4 from total levels of 6.0 +/- 0.3 ng/10(6) cells to zero (mean +/- SD, n = 3). Pretreatment with either cytochalasin B or vinblastine did not alter neutrophil generation of LTB4 induced by calcium ionophore. Neutrophils pretreated with vinblastine, at 4 x 10(-6) to 4 x 10(-4) M, and then maintained at one-half these concentrations during incubation with unopsonized zymosan particles exhibited no diminution in particle ingestion, but were markedly reduced in zymosan-induced synthesis of LTB4.(ABSTRACT TRUNCATED AT 400 WORDS)     

The interaction of lymphocyte surface-bound immune complexes and neutrophils

Neutrophils can be stimulated directly by a variety of stimulants resulting in the production of highly reactive oxygen derivatives. Included in these stimulants are peripheral blood lymphocytes with bovine serum albumin-anti-bovine serum albumin immune complexes (BSA-IC) or aggregated gamma-globulin (AHG) bound to their surface receptors. Through the use of chemiluminescence (CL) studies, we found that B lymphocytes preincubated with AHG stimulated neutrophils to a much greater extent than similarly preincubated T lymphocytes. Preincubated Raji cells (a B lymphoblastoid cell line) were also capable of stimulating neutrophils. We further demonstrated that after periods of mixed incubation with neutrophils, lymphocytes with surface-bound AHG did show an abnormal proliferative response to pokeweed mitogen (PWM), but not to phytohemagglutinin (PHA). This was not due to loss of cell viability, as judged by chromium release cytotoxicity assays and trypan blue exclusion, or to neutrophil enzyme release. The data suggest that neutrophils, stimulated with lymphocyte surface-bound immune complexes, are capable of producing an environment of in vitro oxidant stress. This stress, although not great enough to cause a significant decrease in lymphocyte viability, can cause impaired lymphocyte function. Physiologically, this may relate in the long term to immunologic malfunction observed in patients with high levels of circulating immune complexes.     

A fluorescence assay demonstrating stimulation of phagocytosis by haemolymph molecules of Gallerta mellonella

An in vitro microscopic fluorescence assay determining the phagocytic activity of isolated plasmatocytes of Galleria mellonella is described. It was developed to quantify insect cellular immune reactions. The assay, a modification of a method originally established for vertebrate blood cells, is based on the quenching effect of trypan blue on fluorescein-isothiocyanate-labelled yeast cells. Only ingested yeast cells retain their fluorescence after quenching and can be easily distinguished from adhering ones. The technique is highly reproducible and easy to perform. Using this method a phagocytosis-stimulating effect caused by a haemolymph fraction > 100 kDa isolated from G. mellonella is demonstrated.     

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.     

Fc-gamma- and complement receptor mediated elevation in the cytosolic calcium level in human neutrophils.

The effects of differently opsonized zymosan particles, acting solely at Fc-gamma or at complement receptors or at both, on the level of intracellular calcium ([Ca2+]i) in human neutrophils were studied. A biphasic, long-lasting increase in [Ca2+]i was seen in response to IgG-, C3- and fresh serum-opsonized zymosan particles in the presence of extracellular Ca2+. Unopsonized zymosan, acting mainly at CR3 failed to elevate [Ca2+]i. Addition of 1.4 mM EGTA reduced but did not abolish the rise in [Ca2+]i triggered by opsonized zymosan, indicating that Ca2+ is released from intracellular stores. EGTA changed also the kinetic patterns of Ca(2+)-responses possibly by indirectly affecting the extrusion of Ca2+ in neutrophils.     

Microspectrographic analysis of trypan blue-induced fluorescence in oocytes of the Japanese quail

Summary It was shown that the vital dye trypan blue injected subcutaneously is adsorbed on exogenous yolk and stored in oocytes of Japanese quails. The binding sites of the dye could be visualized by fluorescence microscopy. The spectral distribution of the trypan blue-induced fluorescence emitted by yolk granules was analyzed microspectrographically. The analysis revealed that yolk granules exhibit a deep red fluorescence radiation with a maximum intensity at 670 nm, when blue or green excitation light is used. This fluorescence was exclusively induced by the presence of trypan blue, and not by contaminants of the dye. The fluorescence intensity did not decrease during processing of the tissue throughout the different solvents routinely used in light microscopy, especially after fixation in Heidenhain's fluid, nor did it suffer from pronounced fading during irradiation of the tissue. Model experiments showed that the value of the fluorescence emission maximum was concentration-dependent, and that amounts as little as 5×10^–3 mg trypan blue per ml solution containing an excess of yolk as a substrate for the dye, could clearly be detected and measured.It is suggested that a highly diluted solution of trypan blue can be used without teratogenic effects, as a tracer for exogenous yolk uptake and migration into oocytes, and that fluorescence microscopy is a reliable method for its further localization. A detailed account of the procedure is reported.     

Interaction study between synthetic glycoconjugate ligands and endocytic receptors using flow cytometry

Flow cytometric analysis of synthetic galactosyl polymers, asialofetuin and LDL derivatives labeled with FITC (Fluorescein Isothiocyanate) was carried out to determine the phenotypes of endocytic receptors, such as asialoglycoprotein (ASPG) and the LDL receptor, on various types of cells. When FITC-labeled galactosyl polystyrene (GalCPS), being a synthetic ligand of ASPG, was applied to rat hepatocytes and human cancer cells (Hep G2 and Chang Liver), surface fluorescence intensities varied according to receptor expression on the cells. The fluorescence intensity originates from the calcium-dependent binding of the FITC-labeled GalCPS. Although unaltered by pre-treatment with glucosyl polystyrene (GluCPS), fetuin and LDL, the fluorescence intensity was suppressed by pre-treatment with (non-labeled) GalCPS and asialofetuin. Flow cytometry allowed us to demonstrate that the calcium-dependent binding of FITC-labeled LDL (prepared from rabbits) upon the addition of 17alpha-ethinyl estradiol enhances LDL receptor expression, and the expression is suppressed upon the addition of a monoclonal antibody to the LDL receptor. The binding efficiency based on the combination of FITC-labeled ligands suggests a possible application for the classification of cell types and conditions corresponding to endocytic receptor expression without the need for immuno-active antibodies or radiolabeled substances. Furthermore, the synthetic glycoconjugate (GalCPS) is shown to be a sensitive and useful marker for classification based on cell phenotype using flow cytometry.     

Staining of viable and nonviable myotubes and of myofibrils by the fluorescent dye merocyanine 540

Merocyanine 540 (MC 540) has been reported to interact specifically with excitable plasma membranes in live cells [3]. Here we show that the MC 540 fluorescence staining pattern previously believed to be characteristic of viable myotubes [3] is observed in formaldehyde-fixed cells. In contrast, viable myotubes show an MC 540 fluorescence staining pattern that is characteristic of cell surface staining (no internal structures fluoresce). The specific I-band and H-zone fluorescence of isolated myofibrils is also consistent with the interpretation that the fluorescence patterns previously reported for viable myotubes are in fact characteristic of cells with disrupted plasma membranes. Time-course observations of MC 540 and trypan blue staining of myotubes suggest that when plasma membrane integrity is lost, MC 540 fluorescence can be visualized inside the cell 5-10 min before trypan blue absorbance. Thus the trypan blue viability assay can be misleading when applied to myotubes.     

Staining of Viable and Nonviable Myotubes and of Myofibrils by the Fluorescent Dye Merocyanine 540

Merocyanine 540 (MC 540) has been reported to interact specifically with excitable plasma membranes in live cells [3]. Here we show that the MC 540 fluorescence staining pattern previously believed to be characteristic of viable myotubes [3] is observed in formaldehyde-fixed cells. In contrast, viable myotubes show an MC 540 fluorescence staining pattern that is characteristic of cell surface staining (no internal structures fluoresce). The specific I-band and H-zone fluorescence of isolated myofibrils is also consistent with the interpretation that the fluorescence patterns previously reported for viable myotubes are in fact characteristic of cells with disrupted plasma membranes. Time-course observations of MC 540 and trypan blue staining of myotubes suggest that when plasma membrane integrity is lost, MC 540 fluorescence can be visualized inside the cell 5–10 min before trypan blue absorbance. Thus the trypan blue viability assay can be misleading when applied to myotubes.     

Transmembrane potential changes during phagocytosis in rat alveolar macrophages

Studies were carried out to measure changes in the transmembrane potential of rat alveolar macrophages during exposure of the cells to zymosan particles or to the membrane perturbant, phorbol-12-myristate-13-acetate (PMA), and to determine if changes in membrane potential are related to superoxide anion release. Exposure of the cells to either zymosan or PMA leads to membrane depolarization, which precedes superoxide anion release. Furthermore, the magnitude of the depolarization is dependent upon the concentration of either zymosan or PMA. During exposure of the alveolar macrophages to increasing levels of zymosan, there is an increase in the amount of superoxide released as well as an increase in the magnitude of the depolarization. Incubation of the cells in medium containing 150 mM K+, a medium which causes membrane depolarization, leads to superoxide release from resting cells and a decrease in the amount of superoxide released from cells exposed to zymosan. These results indicate that release of superoxide anion from rat alveolar macrophages is related to membrane depolarization and suggest that the transmembrane potential change may act as a signal to initiate the phagocytotic responses of the cells.     

Suppression of cell-mediated tumor cell lysis and complement-induced cytotoxicity by trypan blue.

Different forms of cell-mediated cytotoxicity were suppressed in the presence of trypan blue. The systems affected included lysis of antibody-coated tumor cells by normal and C. parvum-stimulated mouse peritoneal cells and lysis of allogeneic targets by immune effector cells. The inhibition, measured in a 4-hr 51Cr release assay, was reversible and did not occur in the presence of 30% fetal calf serum or albumin. Binding between effector and target cells through Fc receptors was not affected, and lysis of allogeneic cells was inhibited at the lytic step rather than at the binding step. In contrast, lysis of sensitized erythrocytes was not inhibited by trypan blue, suggesting that lysis of these targets may not involve the steps required in tumor cell lysis. Trypan blue blocked the function of antibody before binding to target cells and also suppressed complement-induced cytolysis. Most individual complement components were susceptible to the inhibitory action of trypan blue. These results reveal an affinity of trypan blue for proteins in general that may be responsible for many of its biologic actions.     

Synchronized human diploid fibroblasts: progression capabilities of a subpopulation that fails to keep pace with the predominant, rapidly dividing cohort of cells

Highly synchronized cultures of HSF-55 human diploid fibroblasts contain subpopulations of cells with intact plasma membranes that do not participate in the parasynchronous division wave. To determine the fate of these laggard cells, cultures were incubated with BrdU for variable periods to label newly replicated DNA in both the readily synchronizable and nonsynchronizable subpopulations. The kinetics of labeling with BrdU were determined with a two-laser flow cytometric technique that did not employ antibody to BrdU, but instead monitored emission of fluorescence from DNA-specific stains that differed in the degree of BrdU-induced quenching of their fluorescence signals. Approximately 90% of the cells rapidly incorporated BrdU and later divided within a 3 hr period. The remaining 10% of the cells, however, were found to reside within a minority subpopulation that maintained the capacity to traverse the cell cycle, but at a greatly reduced rate relative to the progression capacity of the majority of cells. Cells were viably sorted from these cohorts within the synchronized culture, and their kinetic behavior was determined through direct measurement of their growth rates and plating efficiencies. As predicted by the BrdU labeling studies, the sorted cells from the minority, slowly traversing subpopulation divided at a rate that was 30 to 50% lower than that obtained with cells sorted from the readily synchronizable subpopulation. From consideration of the kinetics of entry into S-phase of the majority and minority subpopulations, protocols are described that should allow preparation of relatively pure populations of both early- and late-replicating species of human DNA.     

Quantification of temporary and permanent subpopulations of bull sperm by an optimized SYBR-14/propidium iodide assay.

BACKGROUND: The quality of bull sperm is a key factor in the field of controlled reproduction. Viability-testing is an important aspect of sperm quality definition, especially after cryopreservation where multiple factors such as handling, freeze-thaw cycle, and preservation media, have an impact on the metabolic and functional state of sperm cells. METHODS: We investigated the commonly used SYBR-14/propidium iodide (PI) assay to obtain functional information about sperm-dye and dye-dye interactions. After optimizing filter settings, dye concentrations and incubation times we used these dyes for an interruption free flow cytometric kinetic analysis of a mixture of viable and dead bovine sperm. RESULTS: For the sensitivity of this method and the separation of the different cellular subpopulations fluorescence quenching of SYBR-14 by PI is mainly responsible. Together with a spectral overlap of the two emission spectra of about 5%, even for a wavelength greater than 700 nm, this quenching effect has to be taken into account for a quantitative understanding of the observed fluorescence intensity signals. The fraction of a temporary "intermediate" population to be observed between the viable and dead cells in an SYBR-14/PI-dot-plot diagram becomes greater after stress on the sperm cells caused by cryopreservation. The temporary fraction of "intermediate" cells is maximal at about 6 min after staining and disappears after about 15 min by shifting towards the dead sperm population. The estimation of this "intermediate" population may be a good indicator for handling and storage induced detrimental effects on bovine sperm cells. CONCLUSION: The SYBR-14/PI assay is a fast, reliable and sensitive method to assess the membrane integrity of bull sperm and to separate viable, dead, and "intermediate" sperm subpopulations.     

Phosphatidylinositol 3-phosphate is generated in phagosomal membranes.

Phagocytic cells such as neutrophils and macrophages engulf and destroy invading microorganisms. After internalization, material captured within the phagosomal membrane is destroyed by a complex process of coordinated delivery of digestive enzymes and reactive oxygen species. Several endosomal, lysosomal, and oxidase components expected to participate in these events have recently been shown to bind PtdIns3P, suggesting that this lipid may play a role in this process. We used live, digital fluorescence imaging of RAW 264.7 cells stably expressing either a PtdIns3P binding GFP-PX domain or a GFP-FYVE domain to visualize changes in the levels and subcellular localization of PtdIns3P during phagocytic uptake of IgG-opsonized zymosan particles. Very similar results were obtained using both PtdIns3P probes. The basal distribution of each PtdIns3P probe was partially cytosolic and partially localized to EEA-1-positive endosomal structures. Within about 2-3 min of zymosan attachment and concomitant with the closure of the phagosomal membrane, GFP-positive vesicles moved toward and attached to a localized area of the phagosome. A dramatic, transient accumulation of GFP probe around the entire phagosome rapidly ensued, accompanied by a transient drop in cytosolic GFP fluorescence. The magnitude and timing of this rise in PtdIns3P clearly suggest that it is an ideal candidate for controlling the early stages of phagosomal maturation.     

The transport kinetics of lanthanide species in a single erythrocyte probed by confocal laser scanning microscopy

 A novel method has been developed to visualize and follow the temporal course of lanthanide transport across the membrane into a single living erythrocyte. By means of confocal scanning microscopy and the optical section technique, the entry of lanthanide ions was followed by the fluorescence quenching of fluorescein isothiocyanate (FITC)-labeled membrane and cytosol. From the difference of the quenching kinetics of the whole section and the central area, the time for diffusion through the membrane and the diffusion in the extracellular and intracellular media can be deduced. To clarify the mechanism of lanthanide-induced fluorescence quenching of FITC-labeled erythrocytes and to ensure that this reaction can be used in this method, the reaction was investigated by steady-state fluorescence techniques. The results showed that the lanthanides strongly quenched the florescence emitted by FITC covalently bound to membrane proteins and cytosolic proteins. The static quenching mechanism is responsible for the fluorescence quenching of FITC-labeled proteins by Ln species. The quenching mechanism is discussed on the basis of complex formation. The dependence of fluorescence quenching on both ion size and the total orbital angular momentum L supports the complexation mechanism. The transport time across the membrane is strikingly correlated with Ln species and extracellular concentration. For a given concentration, the transport time of [Ln(cit)₂]^3– is much shorter than that of Ln³⁺, since they enter the cells via the anion channel. This is supported by the inhibition effect of 4,4′-diisothiocyanato-2,2′-stilbenendisulfonate on the transport of [Ln(cit)₂]^3–. On the other hand, the transport of free Ln³⁺ might be attributed to the enhanced permeability of erythrocytes owing to Ln³⁺ binding. These findings strongly demonstrate the existence of the non-internalization mechanism of Ln species uptake by erythrocytes. Received: 7 January 1999 / Accepted: 7 May 1999     

Flow Cytometric Characterization of Bovine Blood Neutrophil Phagocytosis of Fluorescent Bacteria and Zymosan Particles

A Flow Cytometric method for the evaluation of the phagocytic capacity of bovine blood neutrophils is described. The neutrophils were isolated from bovine blood by a one step discontinuous gradient of Percoll. By this technique of isolation, 90 ± 2.8 % (mean ± s) of the granulocytes in the whole blood were recovered. Isolated neutrophils were incubated with FITC labeled S. aureus or zymosan particles in a ratio of 1:20 and 1:10, respectively, and a final serum concentration of 10 %. Phagocytosis was terminated after 15 min and the number of extracellular bacteria or zymosan particles and the percentage of phagocytic granulocytes were registered by Flow Cytometry (FCM). FCM and microscopic studies revealed that eosinophils play a minor role in the phagocytosis of bacteria. The neutrophils were the main population of the granulocytes which were actively phagocytic. Variation among cows in the ability of their blood neutrophils to phagocytize bacteria was evident.