Tag it violet dye

Six million cells were incubated with 1 mL Phosphate Buffered Saline (PBS) that contained 5 µM Tag-IT violet dye (BioLegend, #425101) for 30 min at 37 °C, after which five milliliters of DMEM medium with 5% FBS was added to terminate the reaction, and the cells were centrifuged at 2000 rpm for 5 min. Cells were resuspended in fresh DMEM medium with 5% FBS and seeded into six-well plates at a density of 300,000 cells per well. Forty-eight hours afterwards, the cells were treated with either DMSO, PVD, CIS, or PVD with CIS. Seventy-two hours after treatment, the cells were trypsinized and collected in Falcon tubes (Corning, #352058, Glendale, AZ, USA) in 500 µL fluorescence-activated cell sorting (FACS) buffer (PBS + 5% FBS). Samples were acquired on a Sony (SH800) flow cytometer and analyzed with FlowJo software (version v10.9.0).

Six million cells were incubated with 1 mL Phosphate Buffered Saline (PBS) that contained 5 µM Tag-IT violet dye (BioLegend, #425101) for 30 minutes at 37 °C, after which five milliliters of RPMI medium with 10% FBS was added to terminate the reaction, and the cells were centrifuged at 1200 rpm for 5 minutes. Cells were resuspended in fresh RPMI medium and seeded into six-well plates at a density of 300,000 cells per well. On the following day, the cells were serum-starved for six hours by incubation in serum-free RPMI medium, and then infected with AdSLFN12 or control AdCMV. Twenty-four hours after infection, the medium was replaced with fresh medium. Seventy-two hours after infection, the cells were trypsinized and collected in Falcon tubes (Corning, #352058, Glendale, AZ, USA) in 1000 µL fluorescence-activated cell sorting (FACS) buffer (PBS + 5% FBS). Samples were acquired on a BD FACSymphony A3 flow cytometer (BD) and analyzed with FlowJo software (TreeStar).