粘附频率含量为原位跨交界的分子相互作用，在细胞与细胞界面的动力学分析
Summary
粘附频率检测，用于测量当两个分子相互作用的细胞表面锚定受体 – 配体相互作用的动力学描述。使用一个微管加压作为粘附传感器的人体红血细胞和整合αLβ2和细胞间粘附分子-1受体和配体相互作用，这种机械为基础的检测例证。
Abstract
微管黏附实验是在1998年开发的，衡量的二维（2D）受体-配体结合动力学。检测使用传感器粘附细胞相互作用的分子之一，并提出一个人的红细胞（RBC）的。它采用显微操作与另一个细胞的表达与精确控制的区域和时间，使债券形成的其他相互作用的分子接触，使红细胞。红细胞伸长后发现，除了拉动两个细胞黏附事件。通过控制固定的红细胞表面上的配体的密度，粘连的概率保持在0和1之间的中档。粘连的概率估计在两个细胞之间的一个给定的接触时间的反复接触，周期序列的粘附事件的频率。不同的接触时间，产生一个具有约束力的曲线。拟合概率模型的结合受体-配体反应动力学1曲线返回2D的亲和力和关闭率。
该试剂盒已通过验证，使用与IgG的Fc 段 1-6，选择素与糖缀合物的配体6-9，与配体10-13 homotypical cadherin 的约束力14，T细胞受体与辅助受体肽主要组织相容性复合物15整合Fcγ 受体的相互作用- 19。
该方法已用于生物物理因素，如膜microtopology 5，膜锚2，分子取向和长度为6，承运人刚度9，曲率20，和撞击力20，以及生化因素，量化的二维动力学的规定，如调制的骨架和膜的微环境相互作用的分子， 这些分子表面，15,17,19组织。
该方法也被用来吨Ø研究并发约束力的双种受体-配体3,4，和19三分子相互作用使用修改后的模型 21 。
该方法的主要优点是，它允许在他们的母语膜环境受体的研究。结果可能会非常不同，从获得使用纯化的受体 17 。它还允许在时间分辨率，远远超出了典型的生化方法分一秒的时间尺度的受体 – 配体相互作用的研究。
要说明的微量粘附频率的方法，我们动力学测量间粘附分子-1（ICAM – 1）红细胞约束力的解决方案中的E -选择与二聚体的中性粒细胞激活α大号β2整合素α大号β2功能。
Protocol
1。从全血红细胞隔离准备EAS – 45解决方案。重达从表一的所有成分，并溶于DI水100 200毫升。加水使千毫升解决方案，并调整pH值至8.0。过滤和分装50毫升。冻结储存在-20 ° C。 注：作为一名护士，应当由受过训练的医疗专业等步骤1.2机构审查委员会批准的协议，。 绘制成10毫升含有EDTA管中位数肘静脉3血5毫升，轻轻混匀血用EDTA，立即?…
Discussion
要成功使用微管粘附频率检测应考虑的几个关键步骤。首先，确保记录的具体利益的受体 – 配体系统的相互作用。非特异性控制测量（参见图3，4）保证的特殊性。理想的情况下，非特异性黏附概率应低于0.05，对所有接触的时间段，每个时间点之间的特异性和非特异性黏附概率有显著差异。不同的方法可以用来夫妇的红细胞表面的配体。结果表明，氯化铬耦合方法17了非特异性结合的生物…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项研究是由NIH的拨款R01HL091020，R01HL093723，R01AI077343，并R01GM096187支持。
Materials
| Name of the reagent | Company | Catalogue # | Comments |
|---|---|---|---|
| 10x PBS | BioWhittaker | 17-517Q |
Dilute to 1x with deionized water prior to use |
| Vacutainer EDTA | BD | 366643 | RBCs isolation |
| 10ML PK100 | |||
| Histopaque 1077 | Sigma-Aldrich | 10771 | RBCs isolation |
| Adenine | Sigma-Aldrich | A2786 | EAS-45 preparation |
| D-glucose (dextrose) | Sigma-Aldrich | G7528 | EAS-45 preparation |
| D-Mannitol | Sigma-Aldrich | 6360 | EAS-45 preparation |
| Sodium Chloride (NaCl) | Sigma-Aldrich | S7653 | EAS-45 preparation |
| Sodium Phosphate, Dibasic (Na₂HPO₄) | Fisher Scientific | S374 | EAS-45 preparation |
| L-glutamine | Sigma-Aldrich | G5763 | EAS-45 preparation |
| Biotin-X-NHS | Calbiochem | 203188 | RBCs biotinylation |
| Dimethylformamide (DMF) | Thermo Scientific | 20673 | RBCs biotinylation |
| Borate Buffer (0.1M) | Electron Microscopy Sciences | 11455-90 | RBCs biotinylation |
| Streptavidin | Thermo Scientific | 21125 | Ligand functionalizing |
| BSA | Sigma-Aldrich | A0336 | Ligand functionalizing |
| Quantibrite PE Beads | BD Biosciences | 340495 | Density quantification |
| Flow cytometer | BD Immunocytometry Systems | BD LSR II |
Density quantification |
Capillary Tube 0.7-1.0mm x 30" |
Kimble Glass Inc. | 46485-1 | Micropipette pulling |
| Mineral Oil | Fisher Scientific | BP2629-1 | Chamber assembly |
| Microscope Cover Glass | Fisher Scientific | 12-544-G | Chamber assembly |
PE α-human CD11a Clone HI 111 |
eBioscience | 12-0119-71 | Reagent for Fig.1 |
| PE anti-human CD54 | eBioscience | 12-0549 | Reagent for Fig.1 |
| Mouse IgG1 Isotype Control PE | eBioscience | 12-4714 | Reagent for Fig.1 |
| hydraulic micromanipulator | Narishige | MO-303 | Micropipette system |
| Mechanical manipulator | Newport | 461-xyz-m, SM-13, DM-13 | Micropipette system |
| piezoelectric translator | Physik Instrumente | P-840 | Micropipette system |
| LabVIEW | National Instruments | Version 8.6 | Micropipette system |
| DAQ board | National Instruments | USB-6008 | Micropipette system |
| Optical table | Kinetics Systems | 5200 Series | Micropipette system |
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Tags
Adhesion Frequency AssayIn Situ Kinetics AnalysisCross-junctional Molecular InteractionsCell-cell InterfaceMicropipette Adhesion AssayReceptor-ligand Binding KineticsHuman Red Blood Cell (RBC)MicromanipulationBond FormationAdhesion EventRBC ElongationLigands ImmobilizedAdhesion ProbabilityBinding CurveProbabilistic ModelReceptor-ligand Reaction Kinetics2D AffinityOff-rateFcγ ReceptorsIgG Fc1-6SelectinsGlycoconjugate LigandsIntegrinsHomotypical Cadherin BindingT Cell ReceptorCoreceptorPeptide-major Histocompatibility Complexes
Citazione di questo articolo
Zarnitsyna, V. I., Zhu, C. Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface. J. Vis. Exp. (57), e3519, doi:10.3791/3519 (2011).