Related Experiment Video
Updated: Aug 8, 2026
Method for Measurement of Viral Fusion Kinetics at the Single Particle Level
Published on: September 7, 2009
Human immunodeficiency virus envelope-dependent cell-cell fusion: a quantitative fluorescence cytometric assay
Leonor Huerta1, Edmundo Lamoyi, Armida Báez-Saldaña
1Department of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Distrito Federal, México.
Insights
This study presents a novel fluorescent assay to measure cell fusion, crucial for understanding HIV entry and syncytia formation. The method accurately quantifies fusion extent, aiding research into factors influencing cell-cell fusion processes.
Area of Science:
- Virology
- Cell Biology
- Immunology
Background:
- In vitro cell fusion assays are vital for studying human immunodeficiency virus (HIV) entry mechanisms and syncytia formation.
- These assays typically involve cells expressing HIV envelope proteins (gp120/gp41) and target cells with CD4 and chemokine coreceptors.
Purpose of the Study:
- To develop and validate a novel, quantitative assay for measuring cell-cell fusion.
- To apply this assay to study HIV-glycoprotein-mediated fusion and its inhibition.
Main Methods:
- A new assay was developed using two distinct fluorescent lipophilic probes to label individual cell populations.
- Flow cytometry was employed to quantify the extent of cellular fusion after co-culture.
Main Results:
- Fused cells were identified as double-fluorescent particles, enabling the measurement of fusion proportion.
- The assay determined the kinetics, extent, optimal ratios, and composition of fusion products.
- Inhibition of fusion by soluble CD4 and anti-CXCR4 antibody 12G5 was successfully measured in both monolayer and suspension cell cultures.
Conclusions:
- The developed assay offers simple, minimally invasive, and automatically quantifiable methods for studying cell-cell fusion.
- This technique is valuable for investigating factors that influence fusion in various cell types, including those relevant to HIV infection.
Background:
In vitro fusion of transfected cells expressing the human immunodeficiency virus (HIV) envelope proteins gp120/gp41, with target cells expressing CD4, and a suitable chemokine coreceptor is used widely to investigate the mechanisms of molecular recognition and membrane fusion involved in the entry of the HIV genome into cells and in syncytia formation.
Methods:
We developed an assay that uses two different fluorescent lipophilic probes to single label each reacting cell population and flow cytometry to quantify the extent of cellular fusion after coculture.
Results:
Fused cells are detected as double-fluorescent particles in this assay, therefore permitting measurement of their proportion in the total cell population. The time course and extent of HIV-glycoprotein-related cellular fusion, the optimal cell ratio, the size and cell composition of the fusion products, and the inhibition of fusion caused by soluble CD4 and anti-CXCR4 antibody 12G5 were determined. The assay was applied to measure fusion between gp120/gp41 and CD4-expressing cells growing as monolayers (HeLa/CHO fusion), as well as to suspension lymphocyte cultures (Jurkat/Jurkat fusion).
Conclusions:
The method's simple technical and minimal cell-invasive procedures, as well as its non-ambiguous automatic numerical quantification should be useful for the study of factors influencing cell-cell fusion.
