Summary

检测真正的IgE的表达小鼠B​​系细胞

Published: December 01, 2014
doi:

Summary

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

Abstract

B淋巴细胞免疫球蛋白重链(IgH基因)类别转换重组(CSR)是一种方法,其中最初表达IgM的切换到其他的IgH同种型,如IgA,IgE和IgG抗体。的IgH CSR的体外测定是用于若干生物过程包括从DNA重组和修复的分子和细胞免疫学方面的研究的重要方法, 在体外的CSR测定涉及在活化的B细胞的流式细胞仪测量表面Ig的表达。而IgA和IgG亚类的测量是简单的,IgE介导的通过该方法测定是有问题的,由于可溶性IgE结合到FcεRII/ CD23表达活化的B细胞的表面上。在这里,我们描述的产生IgE的,在文化都发生了CSR小鼠B细胞精确测量一个独特的程序。该方法是基于对在细胞表面的IgE CD23复合物的胰蛋白酶介导的裂解,允许检测由CY产生IgE的B谱系细胞toplasmic染色。这个程序提供了企业社会责任的流式细胞仪分析,IgE的一个方便的解决方案。

Introduction

在免疫球蛋白重链(IGH)类开关重组(CSR)的小鼠和人类, 室内运动场μ恒定区外显子(Cμ)被删除并通过几套下游恒定区外显子的一个(C H S)( 更换Cγ, Cε和Cα),导致从生产的IgM的一个开关来产生其它的Ig类( 例如 IgG抗体,IgE或IgA的)的。 CSR发生内开关(S)的区域,这是位于5'至每一组C H S 1 1-10 kb的序列。激活诱导胞嘧啶核苷脱氨酶(AID)酶通过胞苷脱氨活动启动CSR。

IgE的是过敏性疾病2的关键调解人和IgE水平是如何产生和调控可能会打开大门,新的治疗方法,以过敏性疾病的认识。在小鼠和人类,IgE的是最严格的监管免疫球蛋白同型。是的IgE的水平数千恬正常检测ES小于其他的IgH同种型3,但可以高度升高的疾病状态4。然而,CSR至IgE的是不完全的了解。利用体外激活的B细胞的IL-4与两种抗CD40和LPS结合,诱导的CSR既IgG1和IgE的5。活化的B细胞表达低亲和性IgE受体FcεRII/ CD23 2,6,其结合可溶性IgE的分泌在培养后的CSR。因此,当用流式细胞仪分析,B细胞与受体结合的IgE染色类似于B细胞内源性表达的IgE 7。同时,已知小鼠的B细胞表达低亲和力的IgG受体FcγRIIB1(CD32)8,根据我们的经验它不会出现干扰类别转换为IgG1的测定。然而,B细胞活化培养后测量的IgE切换时,非特异性表面结合的IgE可能掩盖了分析。与普通的染色方法,非IgE表达细胞染色阳性的IgE。

这里所概述的是,已被利用来从小鼠9进行的CSR测定法和检测真正的IgE表达的B细胞的策略– 11。激活B细胞与胰蛋白酶治疗,常见的实验室试剂消化蛋白质,同时删除cytophylic和膜结合表面的IgE。随后的通透和染色质的IgE从而揭示了真正的产生IgE的细胞。

Protocol

注:此处描述的所有实验都按照实验动物管理和使用委员会(IACUC)的指导方针并经动物研究儿童医院(ARCH),马萨诸塞州波士顿。 1.试剂的配制 1000倍的IL-4库存:稀释的IL-4细胞因子,以20微克/毫升的H 2 O或0.1%牛血清白蛋白(BSA)。在200μl等份分配到1.5ml微量管中并储存在-20℃。 1000倍的抗CD40:从制造商获得,在1.0毫克/毫升,保存于4℃。 B细胞刺激介质:向…

Representative Results

这个程序已经成功实施,研究企业社会责任,以IgE的小鼠B细胞。为了证明的效率的测量的CSR,我们刺激的小鼠脾B细胞与抗CD40和IL-4如先前所描述的11。刺激后五天,收集细胞并用上述与荧光标记的IgM,的IgG1,IgE和B220(CD45R)抗体染色的协议处理。门控的爆破淋巴细胞( 图1),的IgE +细胞的明确的人口不能干净地鉴别未经胰蛋白酶处理( 图2a)。然而,之前的固?…

Discussion

小鼠脾B细胞在培养用抗CD40和IL-4的刺激将模拟T辅助2型(T H 2)的相互作用,激励类别转换为IgG1和IgE 5。 B细胞可用于CSR在总脾细胞12或作为纯化的脾B细胞11的情况下被激活。在协议中(步骤2.3)所指出的,B细胞富集是可选的,并且是在实验者的判断,以确定它是否将是有益的。使用CD45R(B220)B细胞的正磁分离标记的珠子在这里使用。分离后,建议通过FACS来检查细…

Disclosures

The authors have nothing to disclose.

Acknowledgements

DRW是由美国国立卫生研究院资助AI089972和AI113217,并通过粘膜免疫学研究团队的支持,并拥有一个事业奖从宝来惠康基金医科大学的科学家。

Materials

Name Company Catalog Number Comments
Anti-Mouse IgE FITC BD Pharmingen 553415 clone R35-72
Rat Anti-Mouse IgG1 PE BD Pharmingen 550083 clone A85-1
Methanol BDH BDH1135-4LP Keep at -20 °C
Falcon Cell Strainer 40 µm Nylon Corning 352340
Falcon Cell Strainer 70 µm Nylon Corning 352350
Anti-Hu/Mo CD45R(B220) PerCP-Cy5.5 eBioscience 45-4052-82 clone RA3-6B2
anti-Mouse/Rat CD40 eBioscience 16-0402-85 clone HM40-3
RPMI Medium 1640 Gibco 11875-093
HEPES (1M) Gibco 15630-080
MEM-NEAA (100X) Gibco 11140-050
Phosphate Buffered Saline (10X) Lifetechnologies (Corning) 46-013-CM
MACS CD45R (B220) microbeads  Miltenyi Biotec 130-049-501
MACS purification column Miltenyi Biotec 130-042-401
IL-4 PeproTech 214-14 Reconstitute in water or 0.1% BSA
Formalin Solution, Neutral Buffered, 10% Sigma Aldrich HT501128-4L
Trypsin-EDTA Solution (10X) Sigma Aldrich T4174-100mL 5.0g Trypsin, 2.0g EDTAŸ4Na per Liter of 0.9% NaCl
Penicillin-Streptomycin Sigma Aldrich P0781-100mL 10,000U Penicillin; 10 mg/mL Streptomycin (100X)
L-Glutamine 200mM Sigma Aldrich G7513
2-mercaptoethanol (99%) Sigma Aldrich M6250-100mL
Red cell lysis buffer Sigma Aldrich R7757
Rat Anti-Mouse IgM RPE/cy7 SouthernBiotech 1140-17 clone 1B4B1
HyClone Fetal Calf Serum Thermo SH30910.03
B cell Stimulation media B cell stimulation media consists of RPMI with fetal calf serum (15%), 20 mM HEPES, 1X MEM-NEAA, 2.0mM L-glutamine,  1X Penicillin-Streptomycin (Penicillin:100 U/ml, Streptomycin: 100 µg/ml), Beta-mercaptoethanol (7 µL/L), IL-4 (20 ng/mL), and anti-CD40 (1 µg/mL)

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Cite This Article
Gallagher, M. P., Shrestha, A., Magee, J. M., Wesemann, D. R. Detection of True IgE-expressing Mouse B Lineage Cells. J. Vis. Exp. (94), e52264, doi:10.3791/52264 (2014).

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