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

Lisa M. Weatherly; Rachel H. Kennedy; Juyoung Shim; Julie A. Gosse

doi:10.3791/50671

JoVE Journal > Immunology and Infection

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Immunologie und Infektion

微孔板的检测，以评估RBL-2H3肥大细胞脱颗粒：三氯生的影响不使用有机溶剂的化学效应

Published: November 01, 2013

doi:

10.3791/50671

Lisa M. Weatherly*^1,2, Rachel H. Kennedy*^1,2, Juyoung Shim¹, Julie A. Gosse^1,2

¹Department of Molecular and Biomedical Sciences,University of Maine, Orono, ²Graduate School of Biomedical Sciences and Engineering,University of Maine, Orono

Summary

肥大细胞脱颗粒，释放过敏介质，过敏，哮喘和寄生虫防御是很重要的。在这里，我们展示了对脱颗粒的药物和毒物的影响评估的^技术，方法，最近利用抗菌剂三氯生^2，表现出强大的抑制作用。

Abstract

肥大细胞在过敏性疾病和寄生虫的免疫防御中扮演着重要的角色。一旦被激活（例如通过过敏原），他们脱颗粒，一个过程，结果在胞吐过敏介质。调制肥大细胞脱颗粒，药物和毒物，可能对人体健康有正面或负面的影响。肥大细胞功能已被详细解剖大鼠嗜碱性白血病肥大细胞（RBL-2H3），使用一种被广泛接受的模型，人类粘膜肥大细胞^3-5。肥大细胞颗粒成分和过敏介质β-氨基己糖苷酶的释放，这是线性地随着组胺从肥大细胞^6，可以容易且可靠地进行测量通过荧光底物的反应，得到衡量的荧光强度在酶标仪测定是服从高通量研究^1。最初发表的NaAl 等^1，我们已经适应了这个脱颗粒检测的筛选f药物和毒物，并在这里展示其使用。

三氯生是一种广谱抗菌剂，是存在于许多消费产品，并已发现在人的皮肤过敏性疾病的^7-11是一种治疗援助，尽管这种效果的机理是未知的。在这里，我们展示了一个检测三氯生对肥大细胞脱颗粒的效果。我们最近发现，三氯生的强烈影响肥大细胞功能^2。在努力避免使用有机溶剂，三氯生与热和搅拌下直接进入含水缓冲液溶解，所得浓度经使用紫外-可见分光光度法_（ε280 = 4200 L / M /厘米^）12。此协议有可能被使用的各种化学品，以确定其对肥大细胞脱颗粒，并更广泛地说，他们的过敏性潜力。

Introduction

肥大细胞高度免疫效应细胞作为主要介质哮喘，过敏，寄生虫国防和癌变^13-16粒。他们居住在几乎所有的血管组织^15，他们在那里安全地存储在细胞质颗粒，直到过敏和炎症介质激活脱颗粒。脱颗粒胞吐作用的膜结合的颗粒，这会导致释放的药理学活性的介质如组胺，类胰蛋白酶和白三烯^15。这个过程的结果我的启动类型是至关重要安装防御寄生虫以及发起过敏，哮喘和致癌反应¹⁵过敏反应。

肥大细胞和嗜碱性粒细胞表达FcεRI受体，高亲和力受体，免疫球蛋白E（IgE）的^17。暴露于变应原或抗原导致聚集多个绑定的IgE的FcεRI受体^17，它是这个S邻-被称为“交联”的IgE结合的Fc受体发起的脱颗粒过程：酪氨酸磷酸化事件，激活磷脂酶C，钙外流，从内部存储，进入细胞内的钙流入¹⁸级联。此钙离子内流所需的脱颗粒，并进一步与膜之前，使颗粒胞吐¹⁵信号颗粒融合。在实验中，钙离子载体可以直接穿过细胞膜^19，它本质上绕过所有的信号转导的钙离子内流步骤²⁰步骤之前，用于穿梭钙，允许通路目标识别为上游或下游有毒物钙信号^20。

的脱颗粒可以迅速和有效地测量通过监测组胺⁶一起从线性的颗粒，释放到细胞上清液中β-氨基己糖苷酶的释放，但我更加容易去检测使用一个简单的酶 – 底物反应，酶标仪测定荧光产物。这微孔板检测，详载于协议部分，是基于一个健壮的方法最初开发的NaAl 等^。1，量化的荧光底物4 -甲基-N-乙酰-β-D-葡萄糖苷的裂解β-氨基己糖苷酶。我们已经修改了药物和毒物检测，以测试效果，三氯生在这里强调。此方法可靠地量化脱颗粒，是一种廉价的替代，例如，流式细胞仪为基础的检测方法^（21），有可能很好地适合各种各样的抗过敏的药物的高通量筛选，以及免疫系统的或过敏的化学物质。根据2007年美国国家研究委员会的报告“在21 ^世纪的毒性测试，最后这一点是特别重要的：愿景和STRAT埃及“（ http://www.nap.edu/openbook.php?record_id=11970 ）的毒理学试验，利用高通量细胞培养，以减少昂贵的使用传统的实验动物，如小鼠的发展，主张。脱粒协议的NaAl 等制定和修改由我们^2，利用RBL-2H3细胞系，这是一个公认的模型对人体黏膜肥大细胞或嗜碱性粒细胞^3-5同源。（RBL-2H3细胞培养的方法在哈钦森等人详述^22）。本试剂盒也有可能适应任何附加的肥大细胞类型。

三氯生（TCS）是一种广谱抗菌药物，已经使用了30年以上，在医院，个人护理产品和消费品^23,24。行动模式为TCS的抗菌特性是抑制脂肪酸的合成，有可能通过抑制烯酰 – 酰基载体蛋白还原酶^25,26。它被发现在全球范围广泛的消费性产品如沐浴露，洗手液，牙膏，漱口水，洗手液，其浓度为0.3％或10 mM的^24。 TCS的广泛使用，导致在人类^27-29河流和溪流³⁰的检测水平。由Allmyr 等人所做的一项研究²⁷表明，TCS和它的代谢产物是存在于血浆和哺乳期的母亲的牛奶。重要的是，TCS很容易被吸收到皮肤^31-37。 Queckenberg 等^37〜12小时内进入人体皮肤吸收10％〜70毫米TCS霜，造成重大浓度在皮肤上，肥大细胞的所在。

TCS已经临床管理人力过敏性皮肤病^7-11，但一直默默无闻的机制，TCS缓解过敏性皮肤病^38。使用荧光酶标仪测定的详细资料D在这段视频中，我们最近表明，在浓度低至2微米，TCS，极大地挫伤了肥大细胞功能和脱颗粒，提供了一个可能的解释这些临床数据^2。除了这些临床数据提供了一个解释，我们的研究结果帕尔默等人^。2表明，TCS目标的下游信号分子钙离子内流。由于钙信号在许多免疫学和其他生物过程的重要性，TCS上的各种必要的生物过程可能会产生不利的影响。事实上，Udoji 等³⁹表明TCS抑制人体自然杀伤细胞溶解活性，另一个重要的先天免疫功能。

除了其潜在的过敏性皮肤病（或反过来说，作为一个immunotoxicant）作为一种辅助治疗帮助，TCS也可能是一种内分泌干扰物^40-49。因此，如何准备这种化学物质在溶液中，我明确的程序s的兴趣毒理学。因为TCS是一个小的疏水性分子，通常用于有机载体，使其更溶于水。在已经过测试，其中TCS的毒性研究，制备涉及在水中溶解的有机溶剂，如乙醇，丙酮，或石油^2,50,51与援助。然而，很多时候这些溶剂具有生物活性，从而解释复杂的化学测试数据^51。事实上，根据到Rufli 等^。52和^53，这是建议的测试解决方案，为水生生物毒性实验准备用物理方法，化学方法，由于化学溶剂创建毒性文物的潜在。我们先前已经表明，TCS溶解在0.24％的乙醇/水（体积/体积），超声处理30分钟挫伤RBL肥大细胞脱颗粒^2。浓度高于0.24％的乙醇溶液，在已被证明能抑制肥大细胞的退化会使nulation ^54,55例有机溶剂毒性研究的潜在混杂因素的影响。

它不仅是重要的考虑溶剂对生物体或细胞用于研究的效果，而且还监测效果的溶剂测试化学品本身是很重要的。例如，Skaare的等⁵¹发现：TCS，溶解在聚乙二醇（通常在牙膏和漱口水）减弱健康女性女性的抗细菌和抗牙斑的作用，而在油中的溶解引起的功能完全丧失。因此，不同溶剂的能力来调节毒物和药物，包括TCS，效果应该考虑在试验设计。油或有香味的添加剂的使用，可能会干扰的影响，在各种产品中的TCS ^50,51。

在努力以消除需要使用有机溶剂，我们改进我们的方法溶解TCS ²时，通过消除使用的有机溶胶发泄。在本协议中，我们直接TCS颗粒溶解成水缓冲热（≤50℃），然后验证的浓度这TCS股票的紫外 – 可见分光光度法。这些改进是可能的，因为TCS是易溶于水高达40μM（ http://www.epa.gov/oppsrrd1/REDs/2340red.pdf ），并已被证明至抗蚀剂的降解，当加热到50℃（呻/ oehha.ca.gov/prop65/public_meetings/052909coms/triclosan/ciba3.pdf ^）56,57。我们也有额外的好处，紫外-可见分光光度法，TCS也被称为摩尔消光系数为4200 L / mol /平方厘米的¹²强烈地吸收280 nm处^58。

到一个缓冲区中的有机溶剂不借助溶解TCS颗粒，该协议提供了一个简单而有效的方法，包括低成本和快速验证浓度，并描述了一个强大的荧光微孔板检测监控化学作用于肥大细胞脱颗粒。

Protocol

请注意，所有的缓冲液配方，包括在一个表中，在结束的协议文本。第1天： 1。细胞的制备规划出96孔板设置方案，围绕测试样品上的布局，以避免边缘效应。分配三个重复每个TCS浓度测试（±脱粒兴奋剂的抗原或载体），以及一式三份自发释放（没有脱颗粒兴奋剂），最大限度地释放（0.2％的Triton X-100 [TX]洗涤剂溶解），以及井保留?…

Representative Results

当加热至50℃下进行90分钟的UV-Vis吸收光谱为TCS产生一个强大的，平滑的曲线之间的260〜300nm的，在280nm处具有峰值，如在图1中示出。紫外-可见分光光度法，因此，一个重要的工具，可以用来计算浓度，既然发布的在280nm处的摩尔吸光系数为4200升/ 12摩尔/厘米。我们已经发现，TCS不会掉出的时间范围内的整个脱颗粒实验的溶液后，50℃加热（数据未示出）。使用这种加热方法直接进入含?…

Discussion

的NaAl 等^[1]在2004年，开发高通量检测脱颗粒肥大细胞生物传感器。它是一个强大的检测，我们已经适应了我们的TCS研究，在这部影片中详述。在此之前的NaAl 等^。1法，肥大细胞脱颗粒已常规通过评估β-氨基己糖苷酶的^59-61，但这些早期的方法，利用一次读取一个样本的荧光。重要的是，的NaAl 等。他们利用高通量的方法样品一读，一时间在荧光酶标仪和以前方法之间?…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

LMW和RHK的缅因大学的研究生院生物医学科学和工程（GSBSE）支持; RHK还得到了缅因州的农业和林业试验场。提供额外拨款，由国家普通医学科学研究所（NIH P20-GM103423），缅因州的农业和林业试验场（批准号ME08004-10，JAG），大学缅因州ADVANCE：涨潮中心（NSF格兰特＃1008498）和研究入门格兰特（JAG）从PhRMA的基础药理/毒理。我们感谢博士。的大卫Holowka和芭芭拉·贝尔德抗原和细胞。雏哈什米，亚历杭德罗·贝莱斯，和安德鲁Abovian的设备和订单的帮助，我们非常感谢。这是缅因州农业和林业试验场出版3311。

Materials

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:
Material Name	Company	Catalogue Number	Comments (optional)
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:
Name	Recipe	Notes
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:
Material Name	Company	Catalogue Number	Type of Plastic
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

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Weatherly, L. M., Kennedy, R. H., Shim, J., Gosse, J. A. A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent. J. Vis. Exp. (81), e50671, doi:10.3791/50671 (2013).

微孔板的检测，以评估RBL-2H3肥大细胞脱颗粒：三氯生的影响不使用有机溶剂的化学效应

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微孔板的检测，以评估RBL-2H3肥大细胞脱颗粒：三氯生的影响不使用有机溶剂的化学效应

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Offenlegungen

Acknowledgements

Materials

Referenzen

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