Analysis of the structure and composition of individual lipoplex particles by flow fluorometry.
A flow fluorometric approach to study cationic lipoid-DNA complexes is presented. The approach uses standard flow cytometry equipment and common fluorescent dyes (BODIPY and ethidium homodimer-2) to detect both lipoid and DNA content in individual particles. In addition, a procedure that allows determination of whether or not liposomes remain intact is described. The procedure is based on monitoring the retention of a polar tracer that has been preloaded into its aqueous compartment. Sample preparation, instrument setup, data analysis, and methodological limitations are described. Applications of the procedure to cationic lipoid-DNA complexes are described, and illustrations are given for the determination of how the lipoid content, composition, and structure of individual lipoplexes in a population evolve over time, starting at about 1 min after DNA and vesicles are mixed. Analogous procedures can be applied to other heterogeneous particles and supramolecular structures.
Pozharski
Edwin V
EV
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208, USA.
Macdonald
Robert C
RC
D001896:Boron Compounds / Q000032:analysis / Q000737:chemistry; D002412:Cations / Q000032:analysis; D004247:DNA / Q000032:analysis*; D004996:Ethidium / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D008055:Lipids / Q000032:analysis*; D008081:Liposomes / Q000737:chemistry
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D002412:Cations; D005456:Fluorescent Dyes; D008055:Lipids; D008081:Liposomes; C018533:ethidium homodimer; D004247:DNA; D004996:Ethidium