L.M.W. and R.H.K are supported by UMaine&#8217;s Graduate School of Biomedical Science and Engineering (GSBSE); R.H.K. was also supported by the Maine Agricultural &#38; Forest Experiment Station. Additional funding was provided by the National Institute of General Medical Sciences (NIH P20-GM103423), the Maine Agricultural &#38; Forest Experiment Station (Grant Number ME08004-10, J.A.G.), the University of Maine ADVANCE Rising Tide Center (NSF Grant # 1008498), and a Research Starter Grant in Pharmacology/Toxicology from the PhRMA foundation (J.A.G.). We thank Drs. David Holowka and Barbara Baird for the antigen and cells. We are grateful to Hina Hashmi, Alejandro Velez, and Andrew Abovian for help with equipment and orders. This is Maine Agricultural &#38; Forest Experiment Station publication number 3311.

Note that all buffer recipes are included in a table at the end of the protocol text.
DAY 1:
1. Preparation of Cells
<ol>
	<li>Plan out 96-well plate setup scheme, centering test samples on the layout in order to avoid edge effects. Allocate three replicates for each TCS concentration tested (&#177; degranulation stimulant of antigen or ionophore), as well as triplicates for spontaneous release (no degranulation stimulant), maximum release (0.2% Triton...

In 2004, Naal et al.1 developed a mast cell biosensor for high-throughput testing of degranulation. It is a robust assay that we have adapted for our TCS studies and detailed in this video. Prior to the Naal et al.1 assay, mast cell degranulation had been routinely assessed via &#946;-hexosaminidase59-61, but these early methods utilized fluorometers in which one sample was read at a time. Importantly, Naal et al. established direct concordance between their mor...

Mast cells are highly granulated immune effector cells that serve as key mediators in asthma, allergies, parasite defense and carcinogenesis13-16. They reside in nearly every vascularized tissue15, where they safely store allergic and inflammatory mediators in cytoplasmic granules until activated to degranulate. Degranulation is the exocytosis of membrane-bound granules, which results in the release of pharmacologically active mediators such as histamine, tryptase, and leukotrienes15. This process results in the initiation of type I hypersensitivity reactions that are critical in mounting defense against parasites as well as initiating allergic, asthmatic, and carcinogenic responses15.
Mast cells and basophils express Fc&#949;RI receptors, the high-affinity receptors for immunoglobulin E (IgE)17. Exposure to an allergen or antigen causes aggregation of multiple IgE-bound Fc&#949;RI receptors17, and it is this so-called "crosslinking" of IgE-bound Fc receptors that initiates the degranulation process: a cascade of tyrosine phosphorylation events, the activation of phospholipase C, efflux of calcium from internal stores, and influx of calcium into the cell18. This calcium influx is necessary for degranulation, and, further, signals granule fusion with the membrane before causing granule exocytosis15. Experimentally, a calcium ionophore can be used to shuttle calcium directly across the cell membrane19, which essentially bypasses all signal transduction steps prior to the calcium influx step20, allowing for the identification of a pathway target by a toxicant as being upstream or downstream of calcium signaling20.
Degranulation can be measured rapidly and effectively by monitoring the release of &#946;-hexosaminidase into cell supernatant, which is released linearly from the granules alongside histamine6, but is much easier to detect using a simple enzyme-substrate reaction and a microplate reader to assay the fluorescent product. This microplate assay, as detailed in the protocol section, is based upon a robust method originally developed by Naal et al.1, which quantifies the cleavage of the fluorogenic substrate 4-methylumbelliferyl-N-acetyl-&#946;-D-glucosaminide by &#946;-hexosaminidase. We have modified the assay to test effects of drugs and toxicants, with triclosan highlighted here. This method reliably quantifies degranulation, is an inexpensive alternative to, for example, flow cytometric-based detection methods21, and has the potential to lend itself nicely to high-throughput screening of a wide variety of anti-allergy drugs, as well as immunotoxic or allergenic chemicals. This last point is particularly important in light of the 2007 National Research Council report "Toxicity Testing in the 21st Century: A Vision and a Strategy" (<a href="http://www.nap.edu/openbook.php?record_id=11970">http://www.nap.edu/openbook.php?record_id=11970</a>), which advocates for the development of high-throughput toxicology tests that utilize cell culture to reduce the costly use of traditional lab animals such as mice. The degranulation protocol developed by Naal et al.1 and modified by us2, utilizes the RBL-2H3 cell line, which is a well-accepted model homologous to human mucosal mast cells or basophils3-5. (Methods for culturing RBL-2H3 cells are detailed in Hutchinson et al.22). This assay could likely be adapted to any attached mast cell type.
Triclosan (TCS) is a broad-spectrum antimicrobial that has been used for more than 30 years in hospitals, personal care products, and consumer goods23,24. The mode of action for TCS&#8217;s antimicrobial characteristic is the inhibition of fatty-acid biosynthesis, likely by inhibiting enoyl-acyl carrier protein reductase25,26. It is found worldwide in a wide range of consumer products such as shower gel, hand lotion, toothpaste, mouthwash, and in hand soaps at concentrations up to 0.3% or 10 mM24. Widespread use of TCS has resulted in detectable levels in humans27-29 and in rivers and streams30. A study done by Allmyr et al.27 demonstrated that TCS and its metabolites are present in both the plasma and milk from nursing mothers. Importantly, TCS is readily absorbed into the skin31-37. Queckenberg et al.37 found ~10% absorption of an ~70 mM TCS cream into human skin within 12 hr, resulting in significant concentration in the skin, where mast cells reside.
TCS has been shown clinically to manage human allergic skin disease7-11, but the mechanism by which TCS alleviates allergic skin diseases has been unknown38. Using the fluorescent microplate assay detailed in this video, we recently demonstrated that TCS, at concentrations as low as 2 &#956;M, significantly dampens mast cell function and degranulation, providing a potential explanation for these clinical data2. In addition to providing an explanation for these clinical data, our findings in Palmer et al.2 suggest that TCS targets signaling molecules downstream of calcium influx. Due to the importance of calcium signaling in many immunological and other biological processes, TCS could potentially have adverse effects on a wide variety of necessary biological processes. In fact, Udoji et al.39 showed that TCS suppresses human natural killer cell lytic activity, another important innate immune function.
Beyond its potential as a therapeutic aid in allergic skin disease (or, conversely, as an immunotoxicant), TCS may also be an endocrine disruptor40-49. Thus, a clear procedure on how to prepare this chemical in solution is of interest to toxicologists. Because TCS is a small hydrophobic molecule, organic vehicles are often used to make it more soluble in water. In most toxicity studies where TCS has been tested, preparation has involved dissolution in water with the aid of an organic solvent such as ethanol, acetone, or oil2,50,51. However, often times these solvents are biologically active themselves, thereby complicating interpretation of the test chemical data51. In fact, according to Rufli et al.52 and others53, it is recommended that test solutions for aquatic toxicity experiments are prepared using physical methods over chemical methods, due to the potential of chemical solvents to create toxicity artifacts. We have previously shown that TCS dissolved in 0.24% ethanol/water (vol/vol) and sonicated for 30 min&#160;dampens RBL mast cell degranulation2. Ethanol at higher concentrations than 0.24% has been shown to dampen mast cell degranulation54,55 -examples of the potentially confounding effects of organic solvents on toxicity studies.
Not only is it important to consider the effect of solvents on the organism or cells used for study, but also it is important to monitor the effect of a solvent on the test chemical itself. For example, Skaare et al.51 found that dissolving TCS in polyethylene glycol (commonly found in toothpastes and mouthwash) weakened anti-bacterial and anti-plaque effects in healthy female women while dissolution in oils caused a complete loss of function. Therefore, the ability of different solvents to modulate toxicant and drug, including TCS, effects should be considered in assay design. Use of oils or flavor additives may interfere with the effects of TCS in various products50,51.
In an effort to eliminate the need to use organic solvents, we improved upon our method for dissolving TCS2 by eliminating the use of an organic solvent. In the present protocol, we dissolve TCS granules directly into aqueous buffer with heat (&#8804;50 &#176;C), and then verify the concentration of this TCS stock by UV-Vis spectrophotometry. These improvements are possible because TCS is soluble in water up to 40 &#181;M (<a href="http://www.epa.gov/oppsrrd1/REDs/2340red.pdf">http://www.epa.gov/oppsrrd1/REDs/2340red.pdf</a>) and has been shown to resist degradation when heated to 50 &#176;C (<a href="http://oehha.ca.gov/prop65/public_meetings/052909coms/triclosan/ciba3.pdf">http://oehha.ca.gov/prop65/public_meetings/052909coms/triclosan/ciba3.pdf</a>)56,57. We also have the added benefit of UV-Vis spectrophotometry, as TCS also is known to strongly absorb at 280 nm58 with a molar extinction coefficient of 4,200 L/mol/cm12.
This protocol provides a simple, yet effective way to dissolve TCS granules into a buffer without the aid of an organic solvent, including low cost and rapid verification of concentration, and describes a powerful fluorescent microplate assay for monitoring chemical effects on mast cell degranulation.

a microplate assay to assess chemical effects on rbl-2h3 mast cell degranulation: effects of triclosan without use of an organic solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

When heated to 50 &#176;C for 90 min, the UV-Vis absorbance spectrum for TCS produces a strong, smooth curve between ~260 and 300 nm, with a peak at 280 nm, as shown in Figure 1. UV-Vis spectrophotometry is, therefore, an important tool that can be utilized to calculate concentration, since the published molar absorption coefficient at 280 nm is 4,200 L/mol/cm12. We have found that TCS does not fall out of solution during the time frame of the entire degranulation experiment, following the 50 &#176;C heating (data...

Watch this Scientific Journal Video about A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent at JoVE.com

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

a-microplate-assay-to-assess-chemical-effects-on-rbl-2h3-mast-cell

Research

JoVE Journal

Immunology and Infection

39.8K vues.  University of Maine, Orono.  Dégranulation des mastocytes, la libération de médiateurs de l'allergie, est important dans les allergies, l'asthme et la défense parasite. Ici, nous démontrons techniques 1 pour évaluer les effets des médicaments et des substances toxiques sur la dégranulation, la méthodologie utilisée récemment pour exposer l'effet inhibiteur puissant agent antibactérien triclosan de 2. 

Vidéo: A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Dilated cardiomyopathy (DCM) is one of the main causes for heart failure in younger adults1. Although genetic disposition and exposition to toxic substances are known causes for this disease in about one third of the patients, the origin of DCM remains largely unclear. In a substantial number of these patients, autoantibodies against cardiac epitopes have been detected and are suspected to play a pivotal role in the onset and progression of the disease2,3. The importance of cardiac autoantibodies is underlined by a hemodynamic improvement observed in DCM patients after elimination of autoantibodies by immunoadsorption3-5. A variety of specific antigens have already been identified2,3 and antibodies against these targets may be detected by immunoassays. However, these assays cannot discriminate between stimulating (and therefore functionally effective) and blocking autoantibodies. There is increasing evidence that this distinction is crucial6,7. It can also be assumed that the targets for a number of cardiotropic antibodies are still unidentified and therefore cannot be detected by immunoassays. Therefore, we established a method for the detection of functionally active cardiotropic antibodies, independent of their respective antigen. The background for the method is the high homology usually observed for functional regions of cardiac proteins in between mammals8,9. This suggests that cardiac antibodies directed against human antigens will cross-react with non-human target cells, which allows testing of IgG from DCM patients on adult rat cardiomyocytes. Our method consists of 3 steps: first, IgG is isolated from patient plasma using sepharose coupled anti-IgG antibodies obtained from immunoadsorption columns (PlasmaSelect, Teterow, Germany). Second, adult cardiomyocytes are isolated by collagenase perfusion in a Langendorff perfusion apparatus using a protocol modified from previous works10,11. The obtained cardiomyocytes are attached to laminin-coated chambered coverglasses and stained with Fura-2, a calcium-selective fluorescent dye which can be easily brought into the cell to observe intracellular calcium (Ca2+) contents12. In the last step, the effect of patient IgG on the cell shortening and Ca2+ transients of field stimulated cardiomyocytes is monitored online using a commercial myocyte calcium and contractility monitoring system (IonOptix, Milton, MA, USA) connected to a standard inverse fluorescent microscope.

The presented method offers a way to detect functional effective cardiotropic autoantibodies in the plasma of patients with dilated cardiomyopathy, irrespective of the specific antigen, by analysing the impact of isolated patient immunoglobulin on cellular shortening and intracellular calcium transients in isolated rat cardiomyocytes.

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs&#8217; mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules&#160;(SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

The goal of the present&#160;protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time&#160;analyses of the secretory granule&#39;s morphology.

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between genetic and environmental factors are known to play an important role in the pathophysiology of atopic dermatitis. However, we still lack a detailed picture of the pathogenesis of this disease. Thus, it is of importance to develop appropriate animal models for elucidating the progression of atopic dermatitis. Moreover, investigating the effect of environmental factors such as air pollutants on atopic dermatitis expands understanding of the disease. Here, we describe a method for inducing atopic dermatitis in rats with neonatal capsaicin treatment and a protocol for exposure of a constant concentration of formaldehyde to rats to reveal effects on the development of atopic dermatitis in infantile and adolescent periods. These protocols have been successfully applied to several experiments and can be used for other substances.

We describe here methods for neonatal capsaicin treatment to induce atopic dermatitis in rats and exposure of vaporized formaldehyde to investigate the effects of formaldehyde inhalation on atopic dermatitis.

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between ...