Immunology and Infection
Published: June 29th, 2016
This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.
Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.
T lymphocyte adhesion mediates a number of distinct processes in a healthy immune system,3 playing critical roles in T cell trafficking and antigen presentation. Whether during immune surveillance or an active immune response these two broad roles for adhesion are critical.4 The physiological signaling events of T cell-endothelial cell interactions are distinct from T cell-antigen presenting cell (APC) interactions, and therefore require distinct methods of study to best understand the signaling cascades involved. The firm adhesion of a T cell to a blood vessel wall during lymphocyte extravasation requires rapid and dynamic integrin activation. T....
1. Plating the CHO-ICAM Cells
Note: The goal of this step is to plate the CHO-ICAM cells in the flow chambers for growth overnight with the goal of generating a confluent monolayer.
Representative results are shown from the flow adhesion assay using Jurkat and primary human CD3+ T cells, as indicated, stimulated with SDF-1α. The negative controls in all shown experiments are unstimulated cells. A threshold basal percent adhesion of unstimulated cells is between 5 - 10%; base adhesion notably above this range indicates a problematic experiment and suggests the start population of T cells were nonspecifically pre-activated in preparation. Fold increase .......
In order to properly analyze T cell adhesion, the stimulant to be included in the study must be considered when choosing an in vitro method. While there are several assays to study signals leading to LFA-1 activation and ICAM-1 binding all methods are not interchangeable. A static adhesion assay10 is best suited to study T cell-APC interactions; alternatively, the shear stress method detailed here is ideal to model T cell-epithelial cell interactions. In vivo, as chemokines are presented alon.......
|T cell samples (cell line or primary)
|Peripheral human T cells
|µ-Slide VI 0.4 ibiTreat
|500 ml glass bottle
|250 ml glass bottle
|Silicone tubing 0.8 mm
|Confocal microscope with incubator chamber
|Any wide field fluorescent microscope
|New Era Pump Systems
|60 ml syringe
|Tissue-culture treated culture dishes
|Trypsin-EDTA (0.25%) Phenol Red
|Heat Inactivated FBS
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