Single-Cell Multiparametric Analysis of Rare HIV-Infected Cells Identified by Duplexed RNAflow-FISH

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.