Fluorescence Leakage Assay to Study Cell-Penetrating Peptide Interaction with Membrane

Abstract

Source: Konate, K. et al. Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide. J. Vis. Exp. (2020)

This video discusses the fluorescence leakage assay to study the interactions of cell-penetrating peptides with cell membranes using cell membrane mimics — large unilamellar vesicles, or LUVs, encapsulated with a fluorescent dye and a quencher.

Protocol

1. Preparation of Large Unilamellar Vesicles (LUVs)

1. Prepare LUVs for their use as cell membrane mimics for fluorescence leakage assay.
2. Mix with a Hamilton glass syringe phosphatidylcholine (DOPC, 786.11 g/mol), sphingomyelin (SM, 760.22 g/mol) and Cholesterol (Chol, 386.65 g/mol) at the molar ratio 4:4:2. The lipid solution is obtained from a stock solution of each lipid solubilized in a methanol/chloroform (3/1; volume/volume) solvent at 25 mg/mL in a 25 mL glass round-bottom flask. Based on 4 µmol of DOPC, 4 µmol of SM, and 2 µmol of Chol, the lipid solution is obtained from stock solution by mixing 126 µL, 117 µL, and ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
25 mL glass round-bottom flask	Pyrex
8-aminonaphthalene-1, 3, 6-trisulfonic acid, disodium salt (ANTS)	Invitrogen	A350	Protect from light
Chloroform	Sigma-Aldrich	288306
Cholesterol	Sigma-Aldrich	C8667
DOPC (dioleoylphosphatidylcholine)	Avanti Polar	850375P	Protect from air
Extruder	Avanti Polar	610000
Fluorimeter	PTI Serlabo
50 µL glass syringe	Hamilton	705N
HEPES	Sigma-Aldrich	H3375
LabAssay Phospholipid	WAKO	296-63801
liquid chromatography column	Sigma-Aldrich
Methanol	Carlo Erba	414902
Nuclepore polycarbonate membrane (0.1 µm pore size, 25 mm diameter)	Whatman	800309
polystyrene cuvette, 10 x 10 x 45 mm	Grener Bio-One	614101
polystyrene semi-micro cuvette, DLS	Fisher Scientific	FB55924
p-xylene-bispyridinium bromide (DPX)	Invitrogen	X1525	Protect from light
quartz fluorescence cuvette	Hellma	109.004F-QS
rotavapor system	Heidolph	Z334898
Sephadex G-50 resin	Amersham	17-0042-01
Sodium azide (NaN3)	Sigma-Aldrich	S2002
Sodium chlorid (NaCl)	Sigma-Aldrich	S5886
Sonicator bath USC300T	VWR	142-6001
Sphingomyelin	Avanti Polar	860062P	Protect from air
Triton X-100	Eromedex	2000-B
Zetaziser NanoZS	Malvern	ZEN3500