Human immunodeficiency virus envelope‐dependent cell‐cell fusion: A quantitative fluorescence cytometric assay

• Published in: Cytometry (New York, N.Y.) N.Y.), 2002-02, Vol.47 (2), p.100-106
• Main Authors: Huerta, Leonor, Lamoyi, Edmundo, Báez‐Saldaña, Armida, Larralde, Carlos
• Format: Article
• Language: English
• Subjects: Animals; CD4 Antigens; Cell fusion; Cell Line; CHO Cells; Cricetinae; Dose-Response Relationship, Immunologic; flow cytometry; Flow Cytometry - methods; fluorescent probes; HeLa Cells; HIV envelope protein gp120; HIV Envelope Protein gp120 - physiology; HIV envelope protein gp41; HIV Envelope Protein gp41 - physiology; Humans; Jurkat Cells; membrane fusion; Membrane Fusion - physiology; Reproducibility of Results; Transfection; viral fusion proteins
• ISSN: 0196-4763; 1097-0320
• DOI: 10.1002/cyto.10051

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. Cytometry 47:100–106, 2002. © 2002 Wiley‐Liss, Inc.