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

Summary

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.

Abstract

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 cells^3-5. Mast cell granule component and the allergic mediator β-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 disease^7-11, 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 ε₂₈₀ = 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.

Introduction

Mast cells are highly granulated immune effector cells that serve as key mediators in asthma, allergies, parasite defense and carcinogenesis^13-16. 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....

Protocol

Note that all buffer recipes are included in a table at the end of the protocol text.

DAY 1:

1. Preparation of Cells

1. 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 (± degranulation stimulant of antigen or ionophore), as well as triplicates for spontaneous release (no degranulation stimulant), maximum release (0.2% Triton X-100 [TX] detergent lysis), as well as wells reserved for background samples (which will contain no cells). For each replicate experimental day, choose a....

Results

When heated to 50 °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 °C heating (data not shown.......

Discussion

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 β-hexosaminidase^59-61, 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RBL-2H3 Cells	ATCC	CRL-2256	The cells we used were a gift, but they are also available from ATCC
Triclosan/Irgasan	Sigma	72779 CAS# 3380-34-5	Should be stored in a low humidity environment
Trypsin	Gibco	25300-054 CAS# 3380-34-5
EMEM	Lonza	12-611F
Fetal Bovine Serum	Atlanta Biologicals	S11150
Gentamycin Sulfate	Lonza Biological Sciences	17-518
Albumin, Bovine Serum	Calbiochem	12659 CAS# 9048-46-8
Surfact-Amps X-100 (Triton X-100; 10% solution)	Pierce	28314 CAS# 9002-93-1
HEPES	J.T Baker	4153-01 CAS# 75277-39-3
Magnesium Chloride	VWR	BDH0244-500G CAS# 7791-18-6
D-(+)-Glucose	Biomedicals	152527 CAS# 50-99-7
Potassium Chloride Crystal	J.T Baker	3046-01 CAS# 7447-40-7
Calcium chloride dihyrdate	Acros Organics	207780010 CAS# 10035-04-8
Glycine	Sigma	G8898 CAS# 56-40-6
4-Methylumbelliferyl-N-acetyl-β-D-glucosaminide (4-MU)	EMD Biosciences	474502-250MG CAS # 37067-30-4	Wrap in foil – is light-sensitive
Anti-DNP Mouse IgE	Sigma	D8406	Reagent has concentration of 1 mg/ml. Aliquot 25 µl of reagent into separate microcentrifuge tubes and Parafilm. Store aliquots at -20 °C that are not being used and store aliquot that is being used at 2-8 °C for no longer than 1 month.
DNP-BSA	Gift from Dr. David Holowka and Dr. Barbara Baird, Cornell University		Suggest: life technologies DNP-BSA catalog# A23018
Calcium Ionophore A23187	Sigma	C75-22-1mg	Ionophore was made from a powder by adding 400 µl of fresh 100% DMSO into the ionophore vial and is kept at -20 °C Note: we have used the ionophore past its 3 month expiration date successfully
DMSO	Sigma	D2650 CAS# 67-68-5
Acetic Acid	VWR	BDH3094-2 CAS# 64-19-7
Anhydrous Sodium Carbonate	Sigma	222321 CAS# 497-19-8
Sodium Chloride	Sigma	71376 CAS# 7647-14-5
Hydrochloric Acid	VWR	BDH3026 CAS# 7647-01-0
Reference Buffer, pH 7	VWR	BDH5046
Reference Buffer, pH 10	VWR	BDH5072
Reference Buffer, pH 4	VWR	BDH5018
pH electrode storage solution	VWR	14002-828
Equipment:
DU 7500 Spectrophotometer	Beckmann	No longer sold
Synergy 2 plate reader Uses Gen5 Microplate Data Collection and Analysis Software	BioTek	Module S
Hematocytometer	Hausser Scientific	3110
7 x 7 CER HOT/STIR 120 V Combination hot plate/magnetic stir plate	VWR	97042-634
Centrifuge	Eppendorf	5430
Tissue culture water bath	VWR	Model# 89032-206
Tissue Culture biological safety cabinet SafeGARD (TC hood)	The Baker Company	Model# SG403A-HE
Tissue culture incubator	ThermoScientific	Model# 3598
Pipetman	VWR		Range: P2-P1000
Balance	Mettler Toledo	Model# AG204
pH meter	Symphony/VWR	Model# SB70P
Pipet-Aid	Drummond Scientific	4-000-100
Combitip dispenser	Eppendorf	4981 000.019
Recipes:
Acetate Buffer, pH 4.4	Make 0.12 M acetic acid and titrate to pH 4.4 with 10 N NaOH. This is 5.3 ml glacial acetic acid into 1 L of MilliQ water: (1 L)*(0.12 mol/L)*(60 g/mol)*(ml/1.37 g) = 5.3 ml because density of glacial is 1.37 g/ml	Sterile Filter into autoclaved glass bottle
Substrate (4-MU)	Sigma M-2133, 250 mg, C18H21NO8, FW 379.4 CAS (37067-30-4) Store in -20°C Stock: 0.12 M in DMSO (46 mg in 1 ml DMSO), warm to 37 °C, vortex, sonicate 10 min. in water-bath sonicator with warm water, vortex again	For each experiment, make fresh solution of substrate in acetate buffer (100x dilution), for final concentration of 1.2 mM in acetate buffer
Glycine Carbonate Buffer, pH 10	26.7 g glycine 47.1 g anhydrous sodium carbonate Add deionized water for 1 L, and adjust pH to 10	Sterile filter into autoclaved glass bottle
Tyrodes (2 L), pH 7.4	135 mM NaCl: 15.78 g (or 270 ml of 1 M) 5 mM KCl: 10 ml of 1 M stock 1.8 mM CaCl2: 7.20 ml of 0.5 M stock 1 mM MgCl2: 4.00 ml of 0.5 M stock 5.6 mM glucose: 2.02 g (11.2 ml of 1 M) 20 mM HEPES: 40 ml of 1 M stock Using concentrated HCl pH from ~9.7-7.4	Sterile filter into autoclaved glass bottle
RBL Cell Media	Thaw fetal bovine serum (FBS, stored at -20 °C) for about 4 hours in 37 °C water bath Follow standard sterile technique Get out 1 L minimum essential medium (MEM) with L-glutamine (with Earle’s salts) Pour off some MEM to have 800 ml MEM, add 200 mL warm FBS Add 1 ml gentamicin sulfate antibiotic to 1 L of media with sterile pipette Only use media bottles that have been autoclaved and marked for cell culture use only.	Sterile filter (0.2 mm) into autoclaved glass bottle
Plastic material used:
200 µl Disposable sterile pipet tips with graduations in 96 rack	VWR	53509-009	polypropylene
1,000 µl Sterile aerosol pipet tips with HighRecovery	VWR	89003-420	polyethylene
10 µl micro tip low binding sterile	VWR	14217-704	polypropylene
Disposable/conical Microcentrifuge tubes for high G-force	VWR	20170-038	polypropylene
Disposable/graduated/conical/sterile 50 ml centrifuge tubes with screw caps	VWR	21008-178	polypropylene
Disposable/graduated/conical/sterile 15 ml centrifuge tubes with screw caps	VWR	21008-103	polypropylene
CELLSTAR Tissue Culture Treated T-25 Flask w/ Filter Cap	Greiner Bio One	690175	polystyrene
CELLSTAR Tissue Culture Treated T-75 Flask w/ Filter Cap	Greiner Bio One	658175	polystyrene
CELLSTAR 10 ml Paper/Plastic Wrapped Serological Pipette	Greiner Bio One	607180	polystyrene
CELLSTAR 2 ml Paper/Plastic Wrapped Serological Pipette	Greiner Bio One	710180	polystyrene
CELLSTAR 5 ml Paper/Plastic Wrapped Serological Pipette	Greiner Bio One	606180	polystyrene
CELLSTAR 25 ml Paper /Plastic Wrapped Serological Pipette	Greiner Bio One	760180	polystyrene
1 cm cuvettes	N/A	N/A	polystyrene
CELLSTAR, 96W Microplate, Tissue-Culture Treated, Black, with Lid 96-well Plate	Greiner Bio One	655086	polystyrene
Combitips	Eppendorf	022266501	Polypropylene/ polyethylene