A high-throughput, microplate reader-based method to monitor in vitro HIV latency reversal in the absence of flow cytometry

Revised Passage:

J-Lat cells, derived from the Jurkat CD4+ T cell line, harbor a non-infectious, inducible HIV provirus tagged with GFP. While these cells have significantly advanced our understanding of HIV latency, their use in many low- and middle-income countries is limited by restricted access to flow cytometry. To address this challenge, we present a modified J-Lat assay that utilizes a standard microplate reader to measure HIV-GFP expression after treatment with latency-reversing agents (LRAs). Our results demonstrate that HIV reactivation by control LRAs, such as prostratin and romidepsin, can be reliably detected with dose-dependent sensitivity, showing strong JNJ-26481585 correlation with flow cytometry. For instance, 10 µM prostratin yielded a 20.1 ± 3.3-fold increase in GFP fluorescence on the microplate reader, corresponding to 64.2 ± 5.0% GFP-positive cells by flow cytometry. Similarly, 0.3 µM prostratin induced a 1.7 ± 1.2-fold increase in fluorescence, matching 8.7 ± 5.7% GFP-positive cells. Using this approach, we screened 79 epigenetic modifiers and identified molibresib, quisinostat, and CUDC-101 as novel LRAs. This microplate reader-based method offers an accessible alternative to flow cytometry, empowering researchers in resource-limited settings to engage in HIV cure research and contribute to global efforts.

Highlights:
- **J-Lat T-cell lines** play a crucial role in HIV cure research but typically require flow cytometry.
- **We introduce an alternative method** using a standard microplate reader to work with J-Lat cells.
- **The assay detects LRAs with comparable sensitivity** to flow cytometry and identifies new LRAs.
- **This method broadens access for resource-limited labs**, enabling more global participation in HIV cure research.