Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer
Published: November 30, 2023
Abstract
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.
Protocol
1. Preparation of Liposomes 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 μM DOPC and 80 μM Biotin-PE phospholipids at 10 ml each, start by placing 629 μl of 10 mg/ml DOPC and 88 μl 10 mg/ml Biotin-PE into separate glass chromatography tub…
開示
The authors have nothing to disclose.
Materials
| 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 |
