Optimization of experimental settings for the analysis of human neutrophils oxidative burst in vitro (original) (raw)
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Evaluation of assays for the measurement of bovine neutrophil reactive oxygen species
Veterinary Immunology and Immunopathology, 2007
During mastitis and other bacterial-mediated diseases of cattle, neutrophils play a critical role in the host innate immune response to infection. Neutrophils are among the earliest leukocytes recruited to the site of infection and contribute to host innate immune defenses through their ability to phagocytose and kill bacteria. The bactericidal activity of neutrophils is mediated, in part, through the generation of reactive oxygen species (ROS). Extracellular release of ROS can induce injury to host tissue as well, and aberrant release of ROS has been implicated in the pathogenesis of certain inflammatory-mediated diseases. Due to their essential role in bacterial clearance and implicated involvement in the pathogenesis of other diseases, there is much interest in the study of neutrophil-generated ROS. Several assays have been developed to measure ROS production, however, many of these have not been evaluated with bovine neutrophils. The objectives of the current study were to evaluate different assays capable of measuring bovine neutrophil ROS, and to compare the results of assays never previously tested with bovine neutrophils to those obtained from more well-established assays frequently used with these cells. Eight different assays were evaluated, including: luminol, isoluminol, and methyl cypridina luciferin analog (MCLA) chemiluminescence assays; Amplex Red, dihydroethidium (DHE), dichlorodihydrofluorescein diacetate (CM-H 2 DCFDA), and dihydrorhodamine 123 fluorescence assays; and the cytochrome c absorbance assay. The assays were evaluated in the context of their abilities to detect ROS produced in response to two agonists commonly used to induce neutrophil activation, phorbol 12-myristate, 13-acetate (PMA) and opsonized zymosan. Diphenyleneiodonium chloride, a NADPH oxidase inhibitor, was used to assess the specificity of the assays to detect ROS. The ability of these assays to discriminate between intra-and extracellular ROS and to specifically detect distinct ROS was evaluated using superoxide dismutase and catalase, which scavenge extracellular superoxide and hydrogen peroxide, respectively. With the exception of the DHE assay, all assays detected bovine neutrophil ROS generation elicited by PMA and zymosan. PMA, but not zymosan, was able to stimulate neutrophil generation of ROS at levels that were detectable with DHE. The MCLA chemiluminescence assay was the only assay that detected ROS produced in response to each of the lowest concentrations of PMA and zymosan tested. To our www.elsevier.com/locate/vetimm Veterinary Immunology and Immunopathology 115 (2007) 107-125
Clinical Biochemistry, 2008
Objectives: To assess the effect of different anticoagulants (EDTA, citrate and heparin) on the isolation procedure of human neutrophils and in the subsequent alterations of calcium levels and respiratory burst induced by phorbol myristate acetate (PMA). Design and methods: Isolation of human neutrophils from whole blood was performed by the gradient density centrifugation method. PMAinduced neutrophil burst was measured by chemiluminescence. Intracellular calcium ([Ca 2+ ] i) was measured using Fluo-3 AM, a calcium-sensitive dye. Results: EDTA provided the highest number of isolated neutrophils/mL of blood (1.7 × 10 6 ± 1.5 × 10 5) when compared with citrate (0.46 × 10 6 ± 0.95 × 10 5) and heparin (0.66 × 10 6 ± 0.15 × 10 5). EDTA originated less degree of PMA-induced activation (370 ± 30%) relatively to citrate (830 ± 98%) and heparin (827 ± 77%). [Ca 2+ ] i was lower with EDTA (122 ± 11 nM) when compared with citrate and heparin (150 ± 13 and 230 ± 30 nM). Conclusion: The anticoagulant used during blood collection interfered differently with the yield of isolated neutrophils as well as on their calcium levels and reactivity to PMA.
Oxidative Metabolism of neutrophils
Polymorphonuclear neutrophils "PMN# have an important role in the host defence response to infection[ These cells produce large amounts of reactive oxygen species "O = − 1 \H 1 O 1 and ONOO − # with microbicidal activity[ PMN are commonly isolated from peripheral blood by sedimentation through a gradient of density "Ficoll!Hypaque gradient and dextran#\ yielding a highly homogeneous cellular population[ However\ some cellular activation due to membrane perturbation is also expected[ We studied how the production of reactive oxygen species and release of myelo! peroxidase "MPO# from blood PMN are a}ected by the use of the Ficoll!Hypaque density gradient[ PMN isolated by spontaneous sedimentation and total blood were used for comparisons[ Lucigenin! and luminol!enhanced chemi! luminescence was used to estimate the production of reactive oxygen from intact cells and shown to be higher for cells isolated by density gradient both in the absence and presence of added stimuli[ The release of MPO\ estimated by the chemiluminescence of the luminol:H 1 O 1 reaction in the supernatant of PMN incubated in the absence and presence of stimuli and absence and presence of cytochalasin B\ was also higher for PMN isolated by a density gradient[ In conclusion\ it was shown that the PMN isolation procedure a}ects reactive oxygen species production and MPO release and in some cases may cause a misinterpretation of results [ Copyright Þ 1999 John Wiley + Sons\ Ltd[ KEY WORDS * oxidative metabolism^myeloperoxidase^polymorphonuclear leukocytes^Ficoll!Hypaque^spontaneous sedi! mentation^blood cells^chemiluminescence ABBREVIATIONS * C\ control^FMLP\ N!formyl!methionyl!leucyl!phenylalanine^MPO\ myeloperoxidase^PMA\ phorbol miristate acetate^PMN\ polymorphonuclear leukocytes^ZY\ opsonized zymosan\ SG\ sedimentation in gradient^SS\ spontaneous sedimentation^TB\ total blood^HRP\ horseradish peroxidase
A Novel Neutrophil Adherence Test Effectively Reflects the Activated State of Neutrophils
Microbiology and Immunology, 1989
We developed a novel and convenient neutrophil adherence test for ascertaining the activated state of neutrophils. Human peripheral blood neutrophils were placed in wells of a 96-well flat-bottom culture plate, and incubated in the presence or absence of neutrophil stimulants for varying periods of time at 37 C in a CO2 incubator (5% CO2, 95% air). After non-adherent cells were completely removed by vibration on a vortex mixer, residual adherent cells were fixed and stained with crystal violet containing 12% formaldehyde and 10% ethanol. After thorough washing, 1% SDS was added to the plate, and the absorbance of each well was measured at 570 nm. Our results correlate well with those of a previously reported method using 51Cr-labeled cells.
The Journal of …, 1984
Mononuclear phagocytes are known to be long-lived cells, capable of synthesis of new cellular constituents and of major alterations in metabolic activities and morphology (1). In contrast, the neutrophil generally has been considered an end-stage cell, incapable of significant functional or metabolic modulation. However, Granelli-Piperno et ai. (2, 3) have reported that human neutrophils actively synthesize protein and RNA and secrete plasminogen activator when placed in culture, and that these activities can be stimulated by incubation of the cells with concanavalin A and inhibited by incubation with glucocorticoids. These findings, and the capacity of small amounts of bacterial endotoxin (lipopolysaccharide, LPS) ~ to prime mononuclear phagocytes for enhanced stimulated release of superoxide anion (O~) (4, 5), suggested to us that neutrophil function might be modified by exposure to LPS. We report here that incubation of human neutrophils for 30-60 min with LPS primes these cells for enhanced release of O~ or hydrogen peroxide (H~O~) upon subsequent contact with a variety of stimuli. This increase in oxidative metabolism might permit increased effectiveness of the neutrophil in host defense. These results also raise the possibility that exposure of neutrophils to bacterial LPS could enhance the capacity of these cells to damage tissue, which might occur in conditions such as endotoxic shock or infection-induced adult respiratory distress syndrome. Materials and Methods Preparation of Neutrophils. Human neutrophils were isolated from venous blood anticoagulated with 10% sodium citrate (3.8% solution in water; Fisher Scientific Co., Pittsburgh, PA). 3 vol blood were added to 1 vol 6% dextran (70,000 tool we) in 0.9% sodium chloride solution (Cutter Laboratories, Berkeley, CA
Comparative Study of Four Isolation Procedures to Obtain Rat Neutrophils
Comparative Clinical Pathology, 2002
The present study was performed to compare four di¡erent methods for isolating neutrophils from rat whole blood. Rat blood has many more lymphocytes than neutrophils when compared to the human blood and this characteristic makes di⁄cult the separation procedure for neutrophils. There are reports in the literature that compare the properties of separation and functional responses of di¡eremt animal species and they usually use Percoll (a solution of silica particles coated with polyvinylpyrrolidone) at di¡erent densities and the in£uence of rat strains on the separation of granulocytes using Percoll solutions has been studied. In this comparative study we used four methods of isolation that use chemical reagents other than Percoll, and a two-step Histopaque gradient technique has been shown to be more appropriate for obtaining rat neutrophils. Many important parameters have been considered for routine employment in making this choice.
The use of flow cytometry to measure neutrophil function
Journal of Immunological Methods, 1999
Neutrophils are important professional phagocytic cells that provide the host with a first line of defense against acute bacterial and fungal diseases and recurrent, severe or unusual infections are associated with inherited defects of neutrophil function. Furthermore, abundant evidence links inappropriate neutrophil-mediated tissue damage to the pathogenesis of conditions such as acute respiratory distress syndrome, septicemia with multiorgan failure, ischemia-reperfusion injury and rheumatoid arthritis. Flow cytometry has been increasingly used to evaluate the functional capabilities of neutrophils. In this review, we discuss the use of flow cytometry to assess neutrophil functional responses including calcium mobilization, Ž . F-actin assembly, adhesion, aggregation, degranulation, phagocytosis and reactive oxygen species ROS production. The use of flow cytometry to identify neutrophil priming is also discussed. The advantage of flow cytometry is that the majority of neutrophil functions can be measured using a small volume of whole blood that reduces artifactual changes in function caused by purification procedures. The advent of numerous new fluorochromes and multiparametric analysis allows the simultaneous measurement of several neutrophil functions in the same population of cells. Flow cytometric analysis provides a rapid screen for abnormalities of neutrophil function and reflects more accurately their behavior in vivo. q
Comparative Study of Neutrophil Activation by Chemiluminescence and Flow Cytometry
Neutrophil activation by phorbol 12-myristate 13-acetate (PMA) and zymosan was assessed by luminol-dependent chemiluminescence (CL) in a novel microtiter plate format and by flow cytometry (FC) based on the oxidation of dihydrorhodamine 123. The results of this comparison demonstrated striking differences in kinetic parameters between these two techniques for neutrophil activation by PMA and zymosan. PMA activation, as determined by FC, was found to be an all-or-none phenomenon in that below a critical concentration of PMA, few cells were positive. Above this concentration, almost all cells were positive; however, the fluorescence intensity of positive cells increased with an increasing PMA concentration until a plateau (maximal) level was reached. In contrast, increasing zymosan concentrations resulted in proportionate increases in the percentage of positive cells until close to 100% of cells were positive. However, the fluorescence intensity of positive cells remained about the same. CL activity increased proportionately with either PMA or zymosan concentration until a maximal level was achieved. The concentration of PMA required for half-maximal activity was about 10-fold higher for FC than for CL, whereas the analogous concentration of zymosan was about 30-fold higher for CL than for FC. In addition, opsonization had only a small negative effect on the ability of zymosan to activate neutrophils, as determined by FC, whereas it had a very large enhancing effect when determined by CL. The differences in kinetic parameters of activation suggest differential sensitivity to particulate (zymosan) versus soluble (PMA) stimulants for FC and CL.