A flow cytometry-based assay to investigate HIV-1 expression in SMYD5 shRNA containing primary CD4+ T cells

Daniela Boehm; Melanie Ott

doi:10.1016/j.xpro.2023.102694

A flow cytometry-based assay to investigate HIV-1 expression in SMYD5 shRNA containing primary CD4⁺ T cells

STAR Protoc. 2023 Dec 15;4(4):102694. doi: 10.1016/j.xpro.2023.102694. Epub 2023 Nov 9.

Authors

Daniela Boehm¹, Melanie Ott²

Affiliations

¹ Gladstone Institute of Virology, University of California San Francisco, San Francisco, CA 94158, USA; Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA.
² Gladstone Institute of Virology, University of California San Francisco, San Francisco, CA 94158, USA; Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA. Electronic address: mott@gladstone.ucsf.edu.

Abstract

Delivering small hairpin RNAs (shRNAs) and the HIV-1 virus to primary CD4+ T cells with high transduction efficiency and high cell viability can be challenging. Here, we present a flow cytometry-based assay to knock down the host protein SMYD5 by shRNA and study the HIV-1 virus specifically in shRNA-containing cells. We describe steps for purifying CD4+ T cells, activating them with CD3/CD28 Dynabeads, transfection of plasmids into HEK293T cells, spinoculation with two different viruses, and analysis by flow cytometry. For complete details on the use and execution of this protocol, please refer to Boehm et al.¹.

Keywords: Cell Biology; Flow Cytometry; Immunology; Molecular Biology.

MeSH terms

CD4-Positive T-Lymphocytes / metabolism
Flow Cytometry
HEK293 Cells
HIV-1* / genetics
Humans
RNA, Small Interfering / genetics

Substances

RNA, Small Interfering

Abstract

MeSH terms

Substances

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