Springer protocols feed by Infectious Diseases 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Measurement of Respiratory Burst Products, Released or Retained, During Activation of Professional Phagocytes
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 consumed O2 is utilized by an NADPH-oxidase to generate highly reactive oxygen species (ROS) by a one electron reduction, initially generating superoxide anion (O2
−) that then dismutates to hydrogen peroxide (H2O2). The ROS are strongly bactericidal molecules but may also cause tissue destruction, and are capable of driving immune competent cells of both the innate and the adaptive immune systems into apoptosis. The developme...
Source: Springer protocols feed by Infectious Diseases - February 28, 2014 Category: Infectious Diseases Source Type: news
Induction and Quantification of Neutrophil Extracellular Traps
Neutrophil extracellular trap (NET) formation is a recently discovered process in the field of innate immunity. It is important to have consistent standards in inducing and quantifying NET formation to compare data from different labs in this new area of investigation. Here we describe the conditions of neutrophil isolation from peripheral blood and stimulation that we find allow the study of NETosis in vitro. The criteria for conclusively identifying the process of NETosis, and the pros and cons of various quantification methods are discussed. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - February 28, 2014 Category: Infectious Diseases Source Type: news
Analysis of Neutrophil Bactericidal Activity
This chapter describes two methods for measuring the bactericidal activity of neutrophils. These are a new simple fluorescence-based assay, which quantifies bactericidal activity by measuring changes in bacterial fluorescence associated with a loss of membrane potential over time, and a more traditional colony counting protocol. Two variations of these techniques are presented: a “one-step” protocol providing a composite measure of phagocytosis and killing, and a “two-step” protocol that allows calculation of separate rate constants for both of these processes. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - February 28, 2014 Category: Infectious Diseases Source Type: news
Quantitative Assessment of Neutrophil Phagocytosis Using Flow Cytometry
Neutrophils have an incredible ability to find and eradicate intruders such as bacteria and fungi. They do this largely through the process of phagocytosis, where the target is internalized into a phagosome, and eventually destroyed by the hostile phagosomal environment. It is important to study phagocytosis in order to understand how neutrophils interact with various pathogens and how they respond to different stimuli. Here, I describe a method to study neutrophil phagocytosis of bacteria using flow cytometry. The bacteria are fluorescently labeled before being introduced to neutrophils. After phagocytosis, both any remai...
Source: Springer protocols feed by Infectious Diseases - February 28, 2014 Category: Infectious Diseases Source Type: news
Expression of Genetically Encoded Fluorescent Probes to Monitor Phospholipid Dynamics in Live Neutrophils
Essential functions of neutrophils, including chemotaxis and phagocytosis, are directed in part by phospholipid signaling. Detailed elucidation of these pathways was hampered by the paucity of methods to study phospholipid localization and dynamics. The development of genetically encoded lipid-specific probes circumvented this limitation. The probes are chimeric constructs consisting of a specific lipid-binding domain fused to a fluorescent protein. This chapter describes a protocol to transiently transfect primary murine neutrophils with such probes in order to localize phospholipids in live cells, and provides a compendi...
Source: Springer protocols feed by Infectious Diseases - February 28, 2014 Category: Infectious Diseases Source Type: news
Immunofluorescence and Confocal Microscopy of Neutrophils
We describe approaches for rapid cell fixation and permeabilization that maximize antigen detection and discuss other variables that also affect data interpretation and image quality (such as cell spreading, degranulation, and phagocytosis). Finally, we show that these methods are also applicable to studies of neutrophil interactions with the extracellular matrix. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - February 28, 2014 Category: Infectious Diseases Source Type: news
Detection of Bidirectional Signaling During Integrin Activation and Neutrophil Adhesion
Neutrophil arrest and migration on inflamed endothelium is dependent upon a conformational shift in CD11a/CD18 (LFA-1) from a low to high affinity and clustered state which determines the strength and lifetime of bond formation with intracellular adhesion molecule 1 (ICAM-1). Cytoskeletal adaptor proteins kindlin-3 and talin-1 anchor clustered LFA-1 to the cytoskeleton and support the transition from neutrophil rolling to arrest. We employ microfluidic flow channels and total internal reflection fluorescence microscopy to evaluate the spatiotemporal regulation of LFA-1 affinity and bond formation that facilitate the transi...
Source: Springer protocols feed by Infectious Diseases - February 28, 2014 Category: Infectious Diseases Source Type: news
Spinning Disk Confocal Imaging of Neutrophil Migration in Zebrafish
Live-cell imaging techniques have been substantially improved due to advances in confocal microscopy instrumentation coupled with ultrasensitive detectors. The spinning disk confocal system is capable of generating images of fluorescent live samples with broad dynamic range and high temporal and spatial resolution. The ability to acquire fluorescent images of living cells in vivo on a millisecond timescale allows the dissection of biological processes that have not previously been visualized in a physiologically relevant context. In vivo imaging of rapidly moving cells such as neutrophils can be technically challenging. In...
Source: Springer protocols feed by Infectious Diseases - February 28, 2014 Category: Infectious Diseases Source Type: news
Neutrophil Migration Through Extracellular Matrix
Neutrophil migration from the bloodstream to sites of infection or injury is a multistep process that requires, dependent on the tissue structures being encountered, different modes of movement. Neutrophil locomotion can range from mesenchymal to amoeboid movement and may include multiple shape changes, contractile squeezing through gaps, and adhesion/de-adhesion cycles. In vitro migration assays reflect only some aspects of the complex in vivo neutrophil recruitment. For two-dimensional in vitro migration chemotaxis chambers, microscopic analysis of movement towards a pipette gradient or Boyden chambers is used. To analyz...
Source: Springer protocols feed by Infectious Diseases - February 28, 2014 Category: Infectious Diseases Source Type: news
Analysis of Electrophysiological Properties and Responses of Neutrophils
We describe the types of measurements that are necessary to characterize a particular ion channel. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - February 28, 2014 Category: Infectious Diseases Source Type: news
Optical Methods for the Measurement and Manipulation of Cytosolic Calcium Signals in Neutrophils
The measurement and manipulation of cytosolic free Ca2+ of neutrophils is crucial for investigating the mechanisms within living neutrophils which generate Ca2+ signals and the cellular responses triggered by them. Optical methods for this are the most applicable for neutrophils and are discussed here, especially the use of fluorescent indicators of Ca2+ and 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, ...
Source: Springer protocols feed by Infectious Diseases - February 28, 2014 Category: Infectious Diseases Source Type: news
Measurement of Phospholipid Metabolism in Intact Neutrophils
Phospholipid-metabolizing enzymes are important participants in neutrophil signal transduction pathways. The methods discussed herein describe assays for assessing the activities of phospholipase A2 (PLA2), phospholipase C (PLC), phospholipase D (PLD), and phosphoinositide 3-OH-kinase in intact neutrophils. PLA2 activity is measured as the release of radiolabeled 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, phosphat...
Source: Springer protocols feed by Infectious Diseases - February 28, 2014 Category: Infectious Diseases Source Type: news
Rho Family and Rap GTPase Activation Assays
The detection of Ras superfamily GTPase activity in innate immune cells is important when studying signaling events elicited by various ligands and cellular processes. The development of high-affinity probes detecting the activated, GTP-bound form of small GTPases has significantly enhanced our understanding of initiation and termination of GTPase-regulated signaling pathways. 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 GTP-bound form of the upstream Rho or Ras GTPase....
Source: Springer protocols feed by Infectious Diseases - February 28, 2014 Category: Infectious Diseases Source Type: news
Generation of Functionally Mature Neutrophils from Induced Pluripotent Stem Cells
Induced pluripotent stem cells (iPSCs) are pluripotent stem cells established from somatic cells. The capability of iPSCs to differentiate into any mature cell lineage under the appropriate conditions allows for modeling of cell processes as well as disease states. Here, we describe an in vitro method for generating functional mature neutrophils from human iPSCs. We also describe assays for testing these differentiated cells for neutrophil characteristics and functions by morphology, cell surface markers, production of reactive oxygen species, microbial killing, and mobilization of neutrophils to an inflammatory site in an...
Source: Springer protocols feed by Infectious Diseases - February 28, 2014 Category: Infectious Diseases Source Type: news
Microinjection Methods for Neutrophils
The ability to microinject substances into the cytosol of living neutrophils opens the possibility of manipulating the chemistry within the cell and also of monitoring changes using indicators which otherwise cannot be introduced into the cell. However, neutrophils cannot be microinjected by the conventional glass pipette insertion method. Here, we outline two techniques which work well with neutrophils, namely, SLAM (simple lipid-assisted microinjection) and electroinjection. (Source: Springer protocols feed by Infectious Diseases)
Source: Springer protocols feed by Infectious Diseases - February 28, 2014 Category: Infectious Diseases Source Type: news
