HIV-1 isolation from infected peripheral blood mononuclear cells

Stefania Dispinseri, Elisa Saba, Elisa Vicenzi, Neeltje A. Kootstra, Hanneke Schuitemaker, Gabriella Scarlatti

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Human immunodeficiency virus 1 (HIV-1) isolation from peripheral blood mononuclear cells (PBMCs) allows retrieval of replication-competent viral variants. In order to impose the smallest possible selective pressure on the viral isolates, isolation must be carried out in primary cultures of cells and not in tumor derived cell lines. The procedure involves culture of PBMCs from an infected patient with phytohemagglutinin (PHA)-stimulated PBMC from seronegative donors, which provide susceptible target cells for HIV replication. HIV can be isolated from the bulk population of PBMCs or after cloning of the cells to obtain viral biological clones. Viral production is determined with p24 antigen (Ag) detection assays or with reverse transcriptase (RT) activity assay. Once isolated, HIV-1 can be propagated by infecting PHA-stimulated PBMCs from healthy donors. Aliquots from culture with a high production of virus are stored for later use.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages187-196
Number of pages10
Volume1087
ISBN (Print)9781627036696
DOIs
Publication statusPublished - 2014

Publication series

NameMethods in Molecular Biology
Volume1087
ISSN (Print)10643745

Keywords

  • Cell culture
  • HIV-1
  • Isolation
  • PBMCs

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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  • Cite this

    Dispinseri, S., Saba, E., Vicenzi, E., Kootstra, N. A., Schuitemaker, H., & Scarlatti, G. (2014). HIV-1 isolation from infected peripheral blood mononuclear cells. In Methods in Molecular Biology (Vol. 1087, pp. 187-196). (Methods in Molecular Biology; Vol. 1087). Humana Press Inc.. https://doi.org/10.1007/978-1-62703-670-2_15