Discriminating in vitro cell fusion from cell aggregation by flow cytometry combined with fluorescence resonance energy transfer.
Expression of fusion proteins in the plasma membrane enables cells to bind and fuse with surrounding cells to form syncytia. Cell fusion can have important functional outcomes for the interacting cells, as syncytia formation does in AIDS pathogenesis. Studies on cell fusion would be facilitated by a quantitative method able to discriminate between cellular aggregates and bona fide fused cells in a cell population. Flow cytometry with fluorescence resonance energy transfer is applied here for analyzing fusion of HIV-1 envelope-expressing cells with CD4+ Jurkat cells. Fusion partners were labeled with the vital lipophilic fluorescent probes DiO (green) and DiI (red) and FRET is manifested by an enhancement of the DiI red fluorescence intensity in double fluorescent cells, thus allowing discrimination between fused and aggregated cells. The inhibitory effect of anti-CD4 monoclonal antibodies and the inhibitory peptide T-20 upon cell fusion were readily quantified by this technique. This method allows the distinction of fused and aggregated cells even when they are at low frequencies.
Huerta
Leonor
L
Department of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad Universitaria, Apartado Postal 70228, Distrito Federal, C.P. 04510, Mexico. leonorhh@biomedicas.unam.mx
López-Balderas
Nayali
N
Larralde
Carlos
C
Lamoyi
Edmundo
E
D015496:CD4-Positive T-Lymphocytes / Q000166:cytology*; D002232:Carbocyanines / Q000032:analysis / Q000494:pharmacology; D002449:Cell Aggregation; D002459:Cell Fusion; D018920:Coculture Techniques; D005434:Flow Cytometry / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D015686:Gene Products, env / Q000096:biosynthesis; D015694:Gene Products, rev / Q000096:biosynthesis; D023581:HIV Fusion Inhibitors / Q000494:pharmacology; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D019169:Jurkat Cells; D013194:Staining and Labeling; D054321:rev Gene Products, Human Immunodeficiency Virus
C035005:3,3'-dihexadecylindocarbocyanine; D002232:Carbocyanines; D005456:Fluorescent Dyes; D015686:Gene Products, env; D015694:Gene Products, rev; D023581:HIV Fusion Inhibitors; D054321:rev Gene Products, Human Immunodeficiency Virus; C098044:3,3'-dioctadecyloxacarbocyanine