eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of CD4+ T Cells for an Image Analysis
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	<li>Thaw a 1 mL aliquot containing 107 frozen peripheral blood mononuclear cells (PBMCs) or lymph node mononuclear cells (LNMCs) from collected samples. In a sterile hood, add the thawed cells to 9 mL of RPMI supplemented with penicillin/streptomycin and glutamine.
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		<li>Cent...

an in vitro technique to study t cell interaction with supported planar lipid bilayers

Source: Steblyanko, M., et al. <a href="https://app.jove.com/v/58143">Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue.</a> J. Vis. Exp. (2018).
This video demonstrates an in vitro technique to study immune synapse formation between primary human T cells and supported lipid bilayers (SLB). T cell surface proteins interact with respective ligands immobilized on SLB to form an immune synapse, followed by the movement of lytic granules towards the synapse, which is visualized using fluorescence microscopy.

An In Vitro Technique to Study T Cell Interaction with Supported Planar Lipid Bilayers

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of CD4+ T Cells for an ...

Take a flow slide containing a supported lipid bilayer, or SLB.
The SLB is conjugated to specific ligands &#8212; intercellular adhesion molecule 1 or ICAM-1 and anti-CD3&#160; antibodies &#8212; labeled with fluorophores.
Add T cells and fluorophore-tagged anti-CD107a Fab &#8212; the antigen-binding fragment of an antibody against the degranulation marker.
The T cell receptor-CD3 or TCR-CD3 complex binds to the anti-CD3 antibody, triggering the TCR.
Triggering-induced inside-out signaling activates integrins on the surface, which bind to the immobilized ICAM-1.
The conjugation induces cytoskeletal reorganization &#8212; microtubule re-arrangement, and actin polymerization that leads to lateral spreading of the cell on the SLB surface.
The spreading allows more ligand-receptor interactions to form a supramolecular activation cluster or SMAC, creating an immune synapse.
T cell lytic granules move along microtubules and fuse with the plasma membrane, exposing degranulation markers on the cell surface, which bind to the anti-CD107a Fab.
Using a fluorescence microscope, visualize the immune synapse enriched in TCR-CD3 complexes, integrins, and degranulation markers.