Abstract
HIV-infected cells are difficult to characterize in vivo because of their great paucity and their diversity. This chapter describes a duplexed flow cytometry method that enables detection, quantification and phenotyping of these rare cells at single-cell resolution. Primary CD4+ T cells are enriched from PBMCs, stained for surface and intracellular proteins and then subjected to fluorescent in situ hybridization to label viral RNA before acquisition on a flow cytometer. Technical and analytical advices are provided to improve the quality of the data. This flow cytometric RNA fluorescent in situ hybridization (RNAflow-FISH) procedure can be applied to the characterization of both HIV-infected cells from viremic people living with HIV and reactivated viral reservoirs from virally suppressed individuals on therapy.
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Acknowledgments
We thank Amy Baxter for the raw data used to exemplify the RNAflow-FISH gating strategy displayed in Fig. 1. We also thank Gérémy Sannier for his insight on the manuscript.
DEK is supported by a FRQS Merit Award (# 268471), the Canadian Institutes of Health Research (CIHR # 152977, CIHR # 164062, CIHR # 168901), and the US National Institutes of Health (NIH UM1-AI-144462 (CHAVD), NIH R01-AI-143411).
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Dubé, M., Kaufmann, D.E. (2022). Single-Cell Multiparametric Analysis of Rare HIV-Infected Cells Identified by Duplexed RNAflow-FISH. In: Poli, G., Vicenzi, E., Romerio, F. (eds) HIV Reservoirs. Methods in Molecular Biology, vol 2407. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1871-4_20
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