eoe sub articles

EoE Sub Articles

1. Preparation of Liposomes
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	<li>Calculate the amount of chloroform-suspended stock solutions of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-cap biotinyl (Biotin-PE) to make diluted stocks at the desired final concentration. To make final concentrations of 400 &#956;M DOPC and 80 &#956;M Biotin-PE phospholipids at 10 ml each, start by placing 629 &#956;l of 10 mg/ml DOPC and 88 &#956;l 10 mg/ml Biotin-PE into separate glass chromatography tub...

super-resolution imaging of nk cell immunological synapse formation on a supported lipid bilayer

Source: Zheng, P., et al. <a href="https://app.jove.com/v/52502">Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer.</a> J. Vis. Exp. (2015).
This video demonstrates a technique for visualizing the formation of the immunological synapse in natural killer (NK) cells on a supported lipid bilayer (SLB). The SLB is first labeled with a fluorophore-tagged NK cell activating receptor ligand, followed by incubation with NK cells. Subsequently, the cells are stained for polymerized actin and perforin-positive lytic granules. This approach enables the visualization of synapse formation using stimulated emission depletion (STED) microscopy.

Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

1. Preparation of Liposomes

	Calculate the amount of chloroform-suspended stock solutions of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and ...

Prepare a chamber slide with a supported lipid bilayer, or SLB, composed of phospholipids and biotinylated phospholipids.
Add blocking proteins to prevent non-specific interactions.
Introduce streptavidin, which binds to the biotinylated phospholipids.
Add biotinylated fluorescently labeled anti-CD16 antibodies, which bind to the immobilized streptavidin.
Pipette D-biotin to prevent non-specific binding of streptavidin to the cells.
Introduce natural killer, or NK, cells. The CD16 receptors on these cells specifically bind to SLB-bound anti-CD16 antibodies, forming an immunological synapse.
This binding triggers actin rearrangement and microtubule polarization, eventually forming microclusters at the synapse center containing SLB-bound anti-CD16 antibodies and CD16 receptors.
Lytic granules containing perforin polarize and penetrate through actin filaments at the synapse.
Fix the cells. Permeabilize the membranes and SLB.
Add fluorescently labeled phalloidin and anti-perforin antibodies, which bind to actin filaments and perforin, respectively.
Using a super-resolution fluorescence microscope, visualize the distribution of fluorescent actin filaments and perforin-positive granules at the synapse.