Methods to monitor liposome fusion, permeability, and interaction with cells.
We describe fluorescence assays for membrane fusion involving the fusion of liposomes with each other and with cultured cells, fluorescence methods to assess liposome uptake by cells and the intracellular delivery of liposome contents, and assays to evaluate liposome membrane permeability. The Tb/DPA and ANTS/DPX assays monitor the intermixing of aqueous contents of liposomes. The NBD-PE/Rhodamine-PE assay follows the intermixing of liposomal lipids. A variation of this method is suitable for detecting the mixing of the inner monolayers of liposomes. The lipid-mixing assay is also used to study the fusion of cationic liposomes and lipoplexes with cultured cells. The intracellular delivery of liposome contents are monitored, via fluorescence microscopy or flow cytometry, by measuring the release of calcein from the liposome interior, and normalized to cell-associated liposomes quantitated with Rhodamine-PE in the membrane of the same liposomes. The release of liposome contents is monitored by the increase in fluorescence of encapsulated carboxyfluorescein, calcein, or ANTS/DPX, or by the decrease in fluorescence of encapsulated Tb/DPA.
Düzgüneş
Nejat
N
Department of Microbiology, University of the Pacific, San Francisco, CA, USA.
Faneca
Henrique
H
Lima
Maria C
MC
D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins; D006801:Humans; D008081:Liposomes / Q000032:analysis* / Q000378:metabolism*; D008561:Membrane Fusion; D008856:Microscopy, Fluorescence / Q000379:methods*; D009281:Naphthalenes; D010539:Permeability; D010848:Picolinic Acids; D011726:Pyridinium Compounds; D013725:Terbium
D005452:Fluoresceins; D008081:Liposomes; D009281:Naphthalenes; D010848:Picolinic Acids; D011726:Pyridinium Compounds; D013725:Terbium; C046466:N,N'-4-xylylenebis(pyridinium); C024098:6-carboxyfluorescein; C067490:terbium chloride; C046465:8-amino-1,3,6-naphthalenetrisulfonic acid; C004860:dipicolinic acid; C007740:fluorexon