竞争的原点实验研究肠嗜T细胞迁移
Summary
竞争归巢实验允许直接评估一个鼠标的两个不同的细胞群的洄游特性。在这里，我们说明了此过程中，通过对比前体内产生的肠道热带与非肠嗜T细胞迁移。
Abstract
为了发挥其功能的淋巴细胞，需要离开血液和迁移到身体的不同组织。淋巴细胞粘附于血管内皮细胞和组织外渗是一个多步骤的过程，不同的粘附分子控制（归巢受体）对淋巴细胞和各自的配体（addressins）1 2内皮细胞上显示。即使这些粘附受体的功能，可部分研究体外，其生理意义的最终测试，以评估他们的作用 ，在体内淋巴细胞的黏附和迁移。两个相辅相成的策略已经用于此目的：活体显微镜（IVM）和归巢的实验。虽然IVM必须确定精确的实时性和在不同组织中的粘附级联在特定的粘附受体的贡献，IVM的是耗费时间和劳动力密集，往往需要复杂的外科技术的发展，它需要同质化的前隔离细胞群，它允许在任何给定的时间只有一个组织/器官的分析。与此相反，有竞争力的归巢实验允许的直接同步比较，在相同的鼠标在两个移民（甚至更多）的细胞亚群，他们还允许许多组织和分析大量的细胞在相同的实验。
在这里，我们描述古典的竞争归巢的协议，用于特定组织，以确定一个给定的细胞类型的优势/缺点相比，控制细胞群。我们选择来说明肠道热带与非肠道嗜T细胞的迁徙，因为肠道黏膜是与外部环境最大的身体接触表面，它也是与最好的定义的迁徙要求额外的淋巴组织。此外，最近的工作已经确定，维生素代谢产物的全反式维甲酸（RA）诱导淋巴细胞的肠道特异性粘附受体（整合a4b7and趋化因子受体CCR9）负责的主要分子机制。因此，我们可以很容易地通过激活T细胞在RA的存在或缺乏肠道热带和非肠道嗜淋巴细胞体外大量产生，分别，终于可以在这里所描述的竞争归巢实验。
Protocol
Discussion
即使归巢的实验提供了非常宝贵的有关在任何特定的组织细胞总人口的迁移的信息，应牢记这些实验不直接分析内皮细胞粘附，因此不歧视的多级粘连步骤（S）级联（圈养/滚动，激活或坚持）一个给定的归巢受体行事。黄金标准中定义的粘附级联的归巢受体的具体的作用，是活体显微镜（IVM），其中个人荧光标记的T细胞（或其他细胞，甚至荧光标记的珠）直接观察与内皮细胞的实时交互。?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
合资是由来自美国S＆结肠炎克罗恩基金会（CCFA）的奖学金支持。 JRM是支持由中国连锁经营协会，癌症研究所（CRI）的，霍华德H.古德曼（MGH），马萨诸塞州的生命科学中心（MLSC）和美国国立卫生研究院主任最新创新奖助学金。
Materials
Animals: C57BL/6mice are commonly used for T cell isolation. In addition, CD45.1 and Thy1.1 congenic strains are available through Jackson Laboratories (Bar Harbor, ME).
Culture media: IMDM (Iscove’s Modified Dulbecco’s Medium + L-Glutamine + Hepes) plus 10% heat-inactivated FBS (Fetal Bovine Serum, low endotoxin, Gibco®, Invitrogen, Carlsbad, CA) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin (HyClone Antibiotics, Waltham, MA), 0.5 mg/ml fungizone/amphotericin B (Gibco), and 50 mM b-mercaptoethanol.
ACK Red Blood Cell Lysis buffer(RBC, 10 mM KHCO3, 150 mM NH4Cl, 0.1 mM EDTA, pH 8.0), adjust to pH 7.2-7.4 and store at room temperature).
PBS (Phosphate Buffered Saline, Hyclone, Waltham, MA).
Flow cytometry (FACS) media(PBS or IMDM + 2% FBS + 5 mM EDTA). When staining using Selectin-Fc chimeras, media with 2 mM Ca++ should be used in all steps (including FACS acquisition). IMDM is recommended in this case.
T cell labeling and adoptive transfer: CFSE(carboxyfluorescein diacetate, succinimidyl ester), CMTMR ((5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine) from Molecular Probes®, Invitrogen, Carlsbad, CA). 1000 x stocks should be made in DMSO (5 mM CFSE, 20 mM CMTMR) and stored at -20°C.
Polyclonal T cell activation: 24-well or 96-well plates (tissue-culture treated, polystyrene, flat-bottom with lid, BD Falcon, Franklin Lakes, NJ) are incubated for 2 hours at 37°C with 50 ml PBS, respectively, containing anti-CD3 plus anti-CD28 antibodies (10 mg/mL each). Then, culture plates are washed twice with PBS and used immediately for T cell culture. Alternatively, Dynabeads coated with anti-CD3/anti-CD28 (Dynal, Invitrogen, Carlsbad, CA) can be used for T cell activation instead of plate-bound antibodies.
Flow cytometry (FACS) staining: Polyclonal activation: CD3 (1452C11), CD28 (37.51). Lineage mAb: CD4 (L3T4), CD8a (Ly-2), Thy1.2 (CD90.2/53-1.2), CD45.2 (104). Gut-homing receptors: purified CCR9 (CD199/eBioCW-1.2, eBioscience, San Diego, CA), a4b7(LPAM-1/DATK3), isotype control (IgG2a, k). Skin-homing receptors: P-selectin-Fc (Purified Mouse P-Selectin – IgG Fusion Protein, BD Pharmingen, San Jose, CA), E-selectin-Fc (Recombinant Mouse E-Selectin/Fc Chimera, R&D Systems, Minneapolis, MN) plus corresponding secondary reagent goat F(ab’)2 anti-human IgG R-PE (Invitrogen, Carlsbad, CA).
All-trans retinoic acid(Sigma, St. Louis, MO)is resuspended in absolute ethanol or DMSO using a yellow bulb or an indirect source of light during the preparation. Store aliquots in glass vials at -80°C and protected from light at all times. Synthetic RAR-agonists: Am80(Wako Chemicals, Richmond, VA).
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