Pi solution

The ratios of intracellular HIV-1 p24⁺ cells, GFP⁺ cells, and the active form of caspase-3 expression were determined as previously described (Hattori et al., 2018 (link); Matsuda et al., 2019 (link)). Briefly, Jurkat/NL or JLTRG/NL cells were washed twice with phosphate buffered salts (PBS) and stained with Ghost Dye Red 780 (TONBO Biosciences, San Diego, CA) for 30 min at 4°C. The cells were then fixed with 1% paraformaldehyde/PBS for 20 min, and permeabilized in a flow cytometry perm buffer (TONBO Biosciences). After 5-min incubation at room temperature (25–30°C), cells were stained with anti-HIV-1 p24 (24-4)-fluorescein isothiocyanate (FITC) monoclonal antibody (mAb; Santa Cruz Biotechnology) and/or Alexafluor 647-conjugated anti-active caspase-3 mAb (C92-605; BD Pharmingen, San Diego, CA) for 30 min on ice. For propidium iodide (PI)/annexin V staining, cells were washed twice with PBS and resuspended in annexin V binding buffer (BioLegend) at a concentration of 1 × 10⁷ cells/mL. The cells were then stained with FITC annexin V (BioLegend) and PI solution (BioLegend) for 15 min at room temperature. Cells were analyzed using a BD FACSVerse flow cytometer (BD Biosciences, Franklin Lakes, NJ). Data collected were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR).

Cells were seeded in 6-well plates (6×10⁵ cells). Under treatment with different drugs for 48 h, 1 mL DNA staining solution was prepared, containing 20 μL PI solution (421301; Biolegend, California, USA), 1 μL Triton X-100 (T9284; sigma, USA), 0.2 μL Rnase A solution (B500474; Sangon Biotech), and PBS (C10010500BT; Life). After the cells were centrifuged at 1000 rpm for 5 min, the cells were resuspended in 100 μl PBS. The cells were fixed with 1 mL 70% ethanol at 4°C overnight. After centrifugation, cells were incubated with 300 μl DNA staining solution at room temperature away from light for 15 min. Cell cycle proportions were detected using CytoFLEX S flow cytometry (Beckman, USA).