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