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 Views.  University of Maine, Orono. 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.