Source: Zheng, P., et al. Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer. 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.
1. Preparation of Liposomes
2. Dialysis of Liposomes
3. Isolating and Culturing Human NK Cells
4. Assembling the Glass-supported Planar Lipid Bilayer
5. Imaging of the NK Synapse on Lipid Bilayer using STED
The authors have nothing to disclose.
18:1 (Δ9-Cis) PC (DOPC) 1,2-dioleoyl-sn-glycero-3-phosphocholine |
Avanti | 850375C | Liposome preparation |
18:1 Biotinyl Cap PE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt) |
Avanti | 870273C | Liposome preparation |
Argon gas, compressed | Airgas | UN1006 | Liposome preparation |
A lyophilizer | Labconco | Freezone 7740020 | Liposome preparation |
Lyophilizer tubes | Labconco | 7540200 | Liposome preparation |
Chromatography Columns | Santa Cruz | sc-205558 | Liposome preparation |
1 M Tris pH 8.0 | Ambion | AM9856 | Liposome preparation |
5 M NaCl | Ambion | AM9759 | Liposome preparation |
Octyl-β-D-glucopyranoside | Sigma-Aldrich | O1008 | Liposome preparation |
Dialysis tubing | Spectrum Labs | 132676 | Liposome preparation |
96-well V-bottom polystyrene plate (untreated) | Corning | 3896 | Antibody density determination |
Non-functionalized silica beads | Bangs Laboratories | SS06N | Antibody density determination |
FACS tubes | Fisher Scientific | 14-959-2A | Antibody density determination |
QuantumTM MESF beads | Bangs Laboratories | Variable | Antibody density determination |
Casein | Sigma-Aldrich | C0875 | Lipid bilayer preparation |
HEPES buffered saline + 1% HSA | Homemade | Lipid bilayer preparation | |
Streptavidin | Life technologies | 434301 | Lipid bilayer preparation |
Fluorescently-labeled biotinylated protein | Homemade | Lipid bilayer preparation | |
ibidi Sticky-Slide VI 0.4 | ibidi | 80608 | Lipid bilayer preparation |
25×75 mm glass coverslip | ibidi | 10812 | Lipid bilayer preparation |
Hydrogen peroxide (30%) | Fisher Scientic | BP2633 | Lipid bilayer preparation |
Sulfuric acid | Sigma-Aldrich | 258105 | Lipid bilayer preparation |
D-Biotin | Invitrogen | B20656 | Lipid bilayer preparation |
Lens paper | VWR | 54826-001 | For imaging |
Type F Immersion Oil | Leica | 11 513 859 | For imaging |
A Leica TCS STED microscope | Leica | For imaging |