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

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記事を引用
Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer. J. Vis. Exp. (Pending Publication), e21845, doi: (2023).

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