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

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

1. 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 tubes.
   NOTE: it is important to clean glass Hamilton syringes and glass chromatography tubes by cleaning solution (1 L 95% ethano....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
25x75 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