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Multivariate analysis techniques were used to separate the effects of various agroecosystem variables on populations of Bemisia tabaci and its native and exotic parasitoids in the Imperial Valley of California. The effects detected were significant and complex. Parasitoid numbers increased with higher B. tabaci density, but positive density-dependence was not observed. Parasitism consistently accounted for a significant portion of B. tabaci mortality, and exotics accounted for a large portion of the mortality clearly attributable to parasitoids especially in alfalfa and broccoli. Percentage parasitism was greatest at sites associated with high regional coverage of alfalfa, and alfalfa seems to be an excellent reservoir for parasitoids. Regional coverage of cantaloupe also seems to enhance biocontrol of B. tabaci at sample sites, including biocontrol from exotics. Populations of parasitoids at sample sites were enhanced by regional coverage of cotton. Populations of parasitoids were lower at sites exposed to relatively high regional insecticide loads, but not necessarily local pesticide loads. This study was initiated only 2 years after the first releases of Eret. sp. nr. emiratus (ex. Ethiopia), the species that eventually became the most numerous exotic parasitoid. It is likely that exotic parasitoid populations will continue to adapt for some time to come, and that their impact has not yet reached its full potential. Once sufficient time has elapsed for populations of introduced species of parasitoids to reach their full potential, multivariate studies of this type can be used determine the impact of natural enemy populations on B. tabaci populations after accounting for the effects of surrounding crops and insecticide use. [ABSTRACT FROM AUTHOR]

Neutrophils are among the first cells to respond to acute inflammation through a multistep process initiated by selectin mediated rolling, which transitions to an integrin/intercellular adhesion molecule-dependent arrest and transmigration across endothelium. A conformational shift in the CD11/CD18 adhesion receptor on neutrophils is a critical determinant of the efficiency of recruitment on inflamed endothelium. For instance, β2-integrin expression level is upregulated up to 10-fold by fusion of cytoplasmic granule pools of CD11b/CD18 (Mac-1). Furthermore, a rapid increase in affinity and membrane clustering of CD11a/CD18 (LFA-1) is necessary for efficient deceleration and arrest in shear flow. We present methods here to quantify the changes in receptor expression and affinity that support neutrophil adhesive phenotypes. Techniques involving real-time fluorescence flow cytometry and parallel plate rheometry coupled with light microscopy are presented. [ABSTRACT FROM AUTHOR]

Neutrophil polarization and directed migration (chemotaxis) are critical for the inflammatory response. Neutrophil chemotaxis is achieved by the sensing of narrow gradients of chemoattractant and the subsequent polarization and directed migration toward the chemotactic source. Despite recent progress, the signaling mechanisms that regulate neutrophil polarization during chemotaxis have not been clearly defined. Here, we describe methods to analyze neutrophil polarization and asymmetric redistribution of signaling components induced by chemoattractant using immunofluorescence. Further, methods are described to dissect the role of specific signaling pathways during chemotaxis by the use of murine neutrophils from transgenic mouse models. Finally, methods for time-lapse microscopy and transwell assay for the analysis of neutrophil chemotaxis will also be discussed. [ABSTRACT FROM AUTHOR]

Polymorphonuclear neutrophils (PMN) represent the dominant cell in the acute response to microbial infection. Effective antimicrobial activity reflects the combined action of soluble agents in plasma with PMN-derived reactive oxygen species and granule proteins, including the azurophilic granule protein myeloperoxidase (MPO). The inhibition or absence of the MPO-H2O2-halide system results in marked reduction in PMN killing of a variety of microbes, implicating its relative prominence in the hierarchy of PMN antimicrobial systems. Although the most profound clinical defects are manifested in patients lacking the capacity to generate reactive oxygen species, as seen in chronic granulomatous disease, an inherited deficiency of MPO can also increase the frequency or severity of clinical infections. As a first step in evaluating the MPO-dependent system, determination of peroxidase activity of isolated leukocytes enriched for PMN coupled with histochemical staining of leukocytes allows assessment of functional MPO within PMN. Immunoblotting of the isolated leukocytes for MPO provides additional information, supplying insight into the molecular basis of the observed absence of functional enzyme. [ABSTRACT FROM AUTHOR]

Inasmuch as neutrophils are the primary cellular defense against bacterial and fungal infections, disorders that affect these white cells typically predispose individuals to severe and recurrent infections. Therefore, diagnosis of such disorders is an important first step in directing long-term treatment/care for the patient. Herein, we describe methods to identify chronic granulomatous disease (CGD), leukocyte adhesion deficiency (LAD), and neutropenia. The assays are relatively simple to perform, cost-effective, and can be performed with equipment available in most laboratories. [ABSTRACT FROM AUTHOR]

Primary disorders of neutrophil function result from impairment in neutrophil responses that are critical for host defense. This chapter summarizes inherited disorders of neutrophils that cause defects in neutrophil adhesion, migration, and oxidative killing. These include leukocyte adhesion deficiencies, actin defects, and other disorders of chemotaxis; hyperimmunoglobulin E syndrome; Chédiak-Higashi syndrome; neutrophil specific granule deficiency; chronic granulomatous disease; and myeloperoxidase deficiency. Diagnostic tests and treatment approaches are also summarized for each neutrophil disorder. [ABSTRACT FROM AUTHOR]

The crucial contribution of neutrophils to innate immunity extends well beyond their traditional role as professional phagocytes. Indeed, it is now well established that neutrophils generate a plethora of inflammatory cytokines and chemokines that are profoundly involved in the onset and evolution of the inflammatory reaction. Several recent studies have also shown that neutrophils can represent an important source of inflammatory cytokines in a number of pathophysiological settings. The inflammatory cytokines produced by neutrophils are generally encoded by immediate-early response genes, which in turn depend on the activation of transcription factors such as those belonging to the nuclear factor (NF)-κB and signal transducers and activators of transcription (STAT) families. We have shown in the past that such factors are expressed and inducible in neutrophils stimulated by physiological agonists. However, the detection of intact (i.e., undegraded) transcription factors in neutrophils requires special precautions and an alternative protocol, as a result of the huge amounts of endogenous proteases present in these cells. This protocol is the focus of this chapter. [ABSTRACT FROM AUTHOR]

In recent years, several studies have brought forward new and exciting discoveries in polymorphonuclear neutrophil research by establishing that the release of inflammatory cytokines constitutes a novel and important aspect of the biology of this cell. At present, neutrophils should no longer be regarded as cells that only release preformed mediators, but instead as fundamental contributors for many events that control and regulate inflammatory, immune, and other relevant responses. In this context, a correct methodological analysis of this novel neutrophil function represents a critical step toward a better understanding of how the release of cytokines by neutrophils may influence pathophysiological processes in vivo. We now describe and discuss methods we have recently developed to more rapidly characterize the pattern of cytokine expression in in vitro-activated human neutrophils. The validation of the real-time PCR assay as a suitable strategy for an accurate, sensitive, reliable, and bona fide analysis of cytokine gene expression in human neutrophils overcomes several problems strictly specific to neutrophils and offers an important tool for high-throughput analysis of gene expression in neutrophils. [ABSTRACT FROM AUTHOR]

Transcriptome analyses of single- and multicellular organisms have changed fundamental understanding of biological and pathological processes across multiple scientific disciplines. Over the past 5 yr, studies of polymorphonuclear leukocyte (or neutrophil) (PMN) gene expression on a global scale have provided new insight into the molecular processes that promote resolution of infections in humans. Herein, we present methods to analyze gene expression in human neutrophils using Affymetrix oligonucleotide microarrays, which include isolation of high-quality RNA, generation and labeling of cRNA, and GeneChip hybridization and scanning. Notably, the procedures utilize commercially available reagents and materials and thus represent a standardized approach for evaluating PMN transcript levels. [ABSTRACT FROM AUTHOR]

The heterodimeric integral membrane protein flavocytochrome b (Cyt b) is the catalytic core of the phagocyte NADPH oxidase, an enzyme complex that initiates a cascade of reactive oxygen species critical for the elimination of infectious agents. Many fundamental questions remain concerning the structure and catalytic mechanism of Cyt b, largely because of the inability to isolate this protein in quantities required for both biochemical analysis and meaningful attempts at high-resolution structure determination. In order to facilitate the direct analysis of Cyt b, the following method describes a rapid and efficient procedure for the immunoaffinity purification of Cyt b (under nondenaturing conditions) from neutrophil membrane fractions. The protocol presented here contains a number of steps that have been optimized and improved since the original description of this Cyt b isolation method. In order to address questions concerning the mechanism of superoxide generation by the NADPH oxidase complex, methods are additionally presented for analysis of conformational dynamics of immunoaffinity-purified Cyt b by resonance energy transfer. [ABSTRACT FROM AUTHOR]

The superoxide (O2•−)-generating enzyme complex of phagocytes, known as the NADPH oxidase, can be assayed in a number of in vitro cell-free (or broken cell) systems. These consist of a mixture of the individual components of the NADPH oxidase, derived from resting phagocytes or in the form of purified recombinant proteins, exposed to an activating agent (or situation), in the presence of NADPH and oxygen. O2•− produced by the mixture is measured by being trapped immediately after its generation with an appropriate acceptor in a kinetic assay, which permits the calculation of the linear rate of O2•− production over time. Cell-free assays are distinguished from whole-cell assays or assays performed on membranes derived from stimulated cells by the fact that all components in the reaction are derived from resting, nonstimulated cells and, thus, the steps of NADPH oxidase activation (precatalytic [assembly] and catalytic) occur in vitro. Cell-free assays played a paramount role in the identification of the components of the NADPH oxidase complex, the diagnosis of various forms of chronic granulomatous disease (CGD), and, more recently, the analysis of the domains present on the components of the NADPH oxidase participating in protein-protein interactions leading to the assembly of the active complex. [ABSTRACT FROM AUTHOR]

Polymorphonuclear neutrophils (PMN) have a remarkable capacity for generation of large amounts of reactive oxygen species in response to a variety of infectious or inflammatory stimuli, a process known as the respiratory burst that involves activation of a multicomponent NADPH oxidase. Given their short life span, PMN are not amenable to most molecular biology methods for studying activation of this oxidant-generating system. We have explored a variety of methods for introduction of components of the phagocytic oxidase (phox system) into the promyelocytic erythroleukemia cell line, K-562. Here, we describe a series of cloned K-562 cell lines that were retrovirally transduced for stable production of one or more essential components of the phagocytic oxidase (phox) complex. We outline methods for the use of these transfectable cells for investigating structure, function, and signaling requirements for assembly and activation of the phox system. These versatile lines can be used to examine effects of genetic polymorphisms or mutations in phox components associated with chronic granulomatous disease, to serve as a system for testing gene therapy vectors designed to correct the defective oxidase, to study cross-functioning with recently described phox component homologs, or to explore signaling components involved in regulation of the respiratory burst. [ABSTRACT FROM AUTHOR]

Activation of professional phagocytes, potent microbial killers of our innate immune system, is associated with an increase in cellular consumption of molecular oxygen (O2). The burst of O2 consumption is utilized by an NADPH-oxidase to generate highly-reactive oxygen species (ROS) starting with one and two electron reductions to generate superoxide anion (O2−) and hydrogen peroxide (H2O2), respectively. ROS are strongly bactericidal but may also cause tissue destruction and induce apoptosis in other immune competent cells of both the innate and the adaptive immune systems. Thus, the development of basic techniques to measure/quantify ROS generation/release by phagocytes during activation of the respiratory burst is of great importance, and a large number of techniques have been used for this purpose. Three of these techniques, chemiluminescence amplified by luminol/ isoluminol, the absorbance change following reduction of cytochrome c, and the fluorescence increase upon oxidation of p-hydroxyphenylacetate, are described in detail in this chapter. These techniques can be valuable tools in research spanning from basic phagocyte biology to more clinically-oriented research on innate immune mechanisms and inflammation. [ABSTRACT FROM AUTHOR]

Using a combination of induced skin blistering and collection chambers permits dynamic studies of the aseptic inflammatory reaction in humans. Blisters filled with interstitial fluid can be generated by applying negative pressure to normal skin for up to 2 h. The blisters are subsequently denuded to form superficial "skin windows" that are well defined with regard to area and depth. The denuded areas are covered with a separate collection chamber filled with a suitable medium and left for 18-24 h. During this period, neutrophils and inflammatory agents accumulate in the chamber medium, and sequential events in the inflammatory process can be studied by repeated sampling. Inactive medium or isolated peripheral blood cells from the same individual can be used as controls for both cellular functions and the pro-/anti-inflammatory mediators that are generated or released. [ABSTRACT FROM AUTHOR]

The primary function of neutrophils is to engulf and destroy invading pathogens. If the bactericidal capacity of neutrophils is defective, an individual may suffer from enhanced susceptibility to potentially fatal microbial infection. To identify such defects, and to investigate the mechanisms used to kill bacteria, the bactericidal activity of neutrophils must be accurately quantified. This chapter provides details of a comprehensive microbiological technique that quantifies neutrophil bactericidal activity by measuring the loss of viability of ingested bacteria over time. Two variations of this technique are presented: a simple "one-step" protocol providing a composite measure of phagocytosis and killing, and a more advanced "two-step" protocol that allows calculation of separate rate constants for both of these processes. [ABSTRACT FROM AUTHOR]

In this chapter, we describe methods to study membrane traffic during phagosome formation and maturation. Although it is convenient to define events as occurring either before (i.e., during formation) or after (during maturation) the creation of a sealed phagosome, it might not be correct to assume that this is reflected by a sudden change in the membrane traffic events involved. Nevertheless, formation events are studied by approaches different from those of maturation events. Before closure of a phagosome, methods relying on immunochemistry and fluorescent markers are employed. Once phagosomes have formed, these can be isolated and additional methods can be used for their characterization. Here, we describe (1) methods to study membrane traffic during phagosome formation and (2) magnetic purification of bacteria-containing phagosomes for maturation studies. [ABSTRACT FROM AUTHOR]

Phagocytosis of invading microorganisms by neutrophils is an important early response to infection. Here, we describe protocols designed for the quantitative study of particle internalization and of the subsequent maturation of the newly formed phagosomes using sheep red blood cells as a model target particle and neutrophils as phagocytes. Techniques to label the particles with fluorescent pH-sensitive dyes are presented, along with complement and immunoglobulin opsonization procedures. These labeling techniques, in combination with high-resolution digital imaging, allow for the quantitative assessment of phagocytosis at the single-cell level by bright field and fluorescence microscopy. Lastly, qualitative and quantitative methods of investigating the intraphagosomal pH are presented. We describe the use of a fluorescent weak base that accumulates in acidic cellular compartments and functions as a marker of phagosome acidification, as well as more quantitative phagosomal pH measurements by fluorescence ratio imaging. A brief description of the hardware and software components necessary for digital imaging is also provided. [ABSTRACT FROM AUTHOR]

Neutrophils are short-lived granulocytes essential for innate host defense. We describe here methods for analysis of resting and activated cells using immunofluorescence and confocal microscopy. Procedures for stimulation of adherent and suspended cells are provided along with protocols for particle opsonization and synchronized phagocytosis. Most importantly, we describe in detail methods for rapid and efficient cell fixation and permeabilization that optimize detection of granule proteins and NADPH oxidase components. Variables that impact antigen detection (such as cell spreading, degranulation, and phagocytosis) are discussed as are methods for image acquisition and analysis. [ABSTRACT FROM AUTHOR]

Polymorphonuclear neutrophil (PMN) recruitment from the blood stream into surrounding tissues, followed by migration through the tissue with triggered release of oxidative enzymes or eventual clearance from the epithelial surface, involves a regulated series of events central to acute responses in host defense (1). Accumulations of large numbers of neutrophils within mucosal tissues are pathognomonic features of both acute and chronic inflammatory conditions including sepsis (2,3) and inflammatory bowel disease (4), but the precise signals governing neutrophil adhesion and transmigration remain to be fully characterized. Previous chapters examine methods employed for both neutrophil isolation and study of the mechanisms underlying regulation of PMN rolling behavior. Here, we describe in vitro experimental models for the examination of PMN adhesion to endothelial and epithelial monolayers as well as the characterization of signals influencing neutrophil migration, both along acellular matrices and across endothelial and epithelial monolayers, in the physiologically relevant directions. Studies employing these model systems allow further elucidation of the mechanisms governing PMN adhesion and transmigration. [ABSTRACT FROM AUTHOR]

Neutrophil migration from blood into tissues is required for effective innate immune responses against infection. Adhesion of the neutrophil in blood to the vascular endothelium and eventual migration through the vessel wall and accumulation at the site of infection involves different classes of adhesion molecules. In vivo intravital microscopy studies show that different adhesion molecules mediate binding events under shear forces associated with blood flow vs binding events that take place under static conditions. To fully analyze the function of these adhesion molecules in vitro, assays must reflect the hemodynamic forces associated with blood flow. We outline two approaches used to study neutrophil adhesion under conditions that mimic blood flow. [ABSTRACT FROM AUTHOR]

We describe here a permeablization method that retains coupling between N-formylmethionyl- leucyl-phenylalanine (fMLP) receptor stimulation and barbed-end actin nucleation in neutrophils. Using fluorescently-tagged actin monomers, we are able to quantify and localize actin-free barbed ends generated downstream of chemoattractant receptors. Partial permeabilization of the neutrophils with the mild detergent n-octyl-β-glucopyranoside maintains signaling from membrane receptor to the actin cytoskeleton while allowing for the introduction of inhibitors and activators of signal transduction pathways implicated in regulating actin cytoskeleton dynamics. This is a useful assay for studying signal transduction to the actin cytoskeleton in neutrophils. [ABSTRACT FROM AUTHOR]

Neutrophil-derived granule enzymes, oxidants, and mediators have been implicated in the pathogenesis of a variety of inflammatory diseases. Neutrophil apoptosis is associated with the loss of expression of adhesion molecules and greatly reduced responsiveness to external stimuli, so that these cells become functionally isolated from their environment. In contrast with necrosis, apoptosis is associated with preservation of plasma membrane integrity, so that release of harmful neutrophil contents is limited, and the inert neutrophils are phagocytosed by local macrophages. Furthermore, phagocytosis of apoptotic neutrophils by human macrophages in vitro suppresses release of macrophage-derived pro-inflammatory mediators. In this way, by downregulating neutrophil functions and triggering "silent" clearance by phagocytes, apoptosis provides a mechanism for the safe disposal of potentially destructive inflammatory cells. Many of the molecular events involved in the apoptosis pathway have been identified and several complementary methods may be employed to identify and quantitate neutrophil apoptosis. This chapter will discuss analysis of neutrophil morphology, DNA fragmentation, membrane changes, mitochondrial alterations, caspase activation, and phagocytosis of apoptotic neutrophils by macrophages. [ABSTRACT FROM AUTHOR]

The past decade has seen increasing use of the patch clamp technique on neutrophils and eosinophils. The main goal of these electrophysiological studies has been to elucidate the mechanisms underlying the phagocyte respiratory burst. NADPH oxidase activity, which defines the respiratory burst in granulocytes, is electrogenic because electrons from NADPH are transported across the cell membrane, where they reduce oxygen to form superoxide anion (O2−). This passage of electrons comprises an electrical current that would rapidly depolarize the membrane if the charge movement were not balanced by proton efflux. The patch clamp technique enables simultaneous recording of NADPH oxidase-generated electron current and H+ flux through the closely related H+ channel. Increasing evidence suggests that other ion channels may play crucial roles in degranulation, phagocytosis, and chemotaxis, highlighting the importance of electrophysiological studies to advance knowledge of granulocyte function. Several configurations of the patch clamp technique exist. Each has advantages and limitations that are discussed here. Meaningful measurements of ion channels cannot be achieved without an understanding of their fundamental properties. We describe the types of measurements that are necessary to characterize a particular ion channel. [ABSTRACT FROM AUTHOR]

The measurement and manipulation of cytosolic free Ca2+ permits the investigation of the mechanisms of generation of the Ca2+ signal and cellular responses to these Ca2+ signals within living neutrophils. The optical methods most applicable to neutrophils, which will be discussed here, are (1) the use of fluorescent indicators of Ca2+ and (2) photoactivation of reagents involved in Ca2+ signaling. Both of these synthetic agents can be loaded into neutrophils as lipid-soluble esters or can be microinjected into the cell. In this chapter, we will outline some of the techniques that have been used to monitor, visualize, and manipulate Ca2+ in neutrophils. [ABSTRACT FROM AUTHOR]

Neutrophils and related phagocytic leukocytes are notoriously difficult to transfect, making the introduction of proteins into these cells for biological studies problematic. We describe here two methods that have been successfully used to introduce proteins into intact primary human neutrophils while maintaining normal functional responses. The first utilizes a lipid-based reagent that transports proteins into intact neutrophils. This method is quick, easy, and is capable of transducing greater than 90% of the neutrophils in the population being studied. The second method involves the addition of a sequence derived from the HIV TAT protein to the protein to be introduced into the neutrophil. This requires both molecular biology to generate the initial construct as well as special procedures for protein isolation and renaturation. However, it also results in highly effective functional protein delivery into human neutrophils. [ABSTRACT FROM AUTHOR]

Polymorphonuclear neutrophils (PMN) play an essential role in host defense against bacteria and fungi through coordinated responses such as adhesion, migration, phagocytosis, secretion, and activation of the NADPH oxidase. The mitogen-activated protein kinases (MAPKs) and their activation kinase cascades, which transduce signals from the plasma membrane to the cytosol and nucleus, are an integral part of signaling pathways involved in many cellular responses. PMN express several members of the MAPK family that have been shown, mainly through the use of pharmacological inhibitors, to mediate the cellular activities triggered by a variety of extracellular agonists. Methods to determine MAPK activation have been greatly simplified with the availability of antibodies raised to active MAPKs. The recent development of novel inhibitors for the MAPK pathways may further our understanding of their role in neutrophil function. [ABSTRACT FROM AUTHOR]

Polymorphonuclear neutrophils play a key role in host defense and inflammation. Neutrophils can be activated by a variety of soluble and particulate factors, leading to an increase in the phosphorylation of numerous proteins on tyrosines, serines and threonines. Upon covalent binding of phosphates to these amino acids, the charge and conformation of the corresponding proteins are modified, generally leading to changes in protein functions such as protein-protein interactions and enzymatic activity. Protein phosphorylation in neutrophils can be studied by following protein incorporation of radiolabeled inorganic phosphate. This technique is based on labeling the intracellular ATP pool with 32P prior to applying a stimulus that induces changes in protein phosphorylation status. The proteins are extracted from the cells, immunoprecipitated or not, resolved on polyacrylamide gel electrophoresis, then transferred onto nitrocellulose membranes and visualized by means of autoradiography. Phosphorylated sites of a multisite-phosphorylated protein can be analyzed by using two-dimensional tryptic phosphopeptide mapping. This chapter describes a phosphorylation protocol and the analysis of the phosphorylation of a neutrophil protein, p47phox, by two-dimensional tryptic phosphopeptide mapping. [ABSTRACT FROM AUTHOR]

Phospholipid metabolizing enzymes are important participants in neutrophil signal transduction pathways. The methods discussed herein describe assays for assessing the activities of phospholipase (PL)A2, PLC, PLD, and phosphoinositide 3-OH-kinase (PI3-K) in intact neutrophils. PLA2 activity is measured as the release of radiolabed arachidonic acid. PLC activity is measured as the accumulation of inositol 1,4,5-trisphosphate (IP3), a water-soluble product, using a commercially available radioreceptor assay kit. PLD activity is measured as the appearance of its radiolabeled products, phosphatidic acid and phosphatidylethanol. PI3-K activity is measured as the appearance of its radiolabeled product, phosphatidylinositol-3,4,5-trisphosphate. [ABSTRACT FROM AUTHOR]

The detection of Ras superfamily GTPase activity in neutrophils is important when studying signaling events elicited by various ligands and cellular processes. Substantial progress in monitoring GTPase activation has been made in recent years by the development of high-affinity probes for small GTPases. These probes are created by fusing a high-affinity GTPase-binding domain derived from a specific downstream effector protein to glutathione-S-transferase (GST). Such domains bind preferentially to the GTPbound form of specific Rho or Ras GTPases. Coupling these probes to beads enables extraction of the complex and subsequent quantification of active GTP-binding protein by immunoblotting. Although effector domains that discriminate efficiently between GDP- and GTP-bound states and highly specific antibodies are not yet available for many small GTPases, analysis of certain members of the Rho and Ras GTPase family are now routinely performed. Here, we describe affinity-based pull-down assays for detection of Rac/Cdc42 and Rap activity in stimulated neutrophils. [ABSTRACT FROM AUTHOR]

The neutrophil has long been recognized for its impressive number of cytoplasmic granules that harbor proteins indispensable for innate immunity. Analysis of isolated granules has provided important information on how the neutrophil grades its response to match the challenges it meets on its passage from blood to tissues. Nitrogen cavitation was developed as a method for disruption of cells on the assumption that sudden reduction of the partial pressure of nitrogen would lead to aeration of nitrogen dissolved in the lipid bilayer of plasma membranes. We find that cells are broken by the shear stress that is associated with passage through the outlet valve under high pressure, and that this results in disruption of neutrophils while leaving granules intact. The unique properties of Percoll as a sedimentable density medium with no inherent tonicity or viscosity are exploited for the creation of continuous density gradients with shoulders in the density profile created to optimize the physical separation of granule subsets and light membranes. Immunological methods (sandwich enzyme-linked immunosorbent assays) are used for quantitation of proteins that are characteristic constituents of the granule subsets of neutrophils. [ABSTRACT FROM AUTHOR]

Advances in the understanding of neutrophil biochemistry require the development of effective procedures for isolating purified neutrophil populations. Although methods for human neutrophil isolation are now standard, similar procedures for isolating neutrophils from many of the nonhuman species used to model human diseases are not as well developed. Because neutrophils are reactive cells, the method of isolation is extremely important to avoid isolation technique-induced alterations in cell function. We present methods here for reproducibly isolating highly-purified neutrophils from large (cow, horse, sheep) and small (mouse, rabbit) animal models and describe optimized details for obtaining the highest cell purity, yield, and viability. We also describe methods to verify phagocytic capacity in the purified cell populations using a flow cytometry-based phagocytosis assay. [ABSTRACT FROM AUTHOR]

Venous blood provides a ready source of large numbers of unstimulated granulocytes and mononuclear cells. Exploiting the differences in the relative densities of the leukocytes circulating in venous blood, one can separate leukocytes from erythrocytes as well as isolate the individual leukocyte populations in high purity for use in ex vivo studies [ABSTRACT FROM AUTHOR]

This valuable and unique book contains a compendium of methods and reviews that does much more than allow one to study the biology of neutrophils. What makes this collection of contributions so special is that it highlights and facilitates using the neutrophil as a simple, pure, single primary cell suspension model to study a remarkable array of generalized cellular functions (ameboid cellular motility, chemotaxis, adhesion, phagocytosis, degranulation, oxygen radical production, apoptosis), biochemical pathways (G protein-coupled receptor function and regulation, ion channel function, calcium transients, phosphorylation events, actin regulation, adhesion molecule regulation), as well as specialized functions and molecules important to host defense against infection, the mediation and resolution of inflammation, and the chemokine mediated modulation of acquired immunity (seeFig. 1). As I note the array of chapter topics, it evokes some of the past history of inquiry into how the neutrophil functions and how we evolved into the current widespread use of the neutrophil as a convenient model system for studying so many types of cellular processes and biochemical pathways. [ABSTRACT FROM AUTHOR]