检测真正的IgE的表达小鼠B系细胞
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）（ 如更换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
Representative Results
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…
Declarações
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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