Ctv dye

An untreated, flat-bottom, 96-well plate was coated overnight at 4°C with 0.2 μg/mL anti-CD3 (clone 145-2C11, BD Biosciences) and 0.5 μg/mL anti-CD28 (clone 37.51, BD Biosciences) in PBS, washed with PBS, and blocked with coculture media (RPMI from Gibco containing 10% FBS from Atlanta Biologicals, 1% penicillin/streptomycin from Gibco, and 1× β-mercaptoethanol from Gibco) for at least 30 minutes at room temperature (RT). CD8⁺ T cells were isolated from the spleens of naive C57BL/6 mice using the untouched CD8⁺ T cell isolation kit (Miltenyi Biotec), following manufacturer’s instructions. Isolated CD8⁺ T cells were washed twice with PBS and stained with CTV dye (Life Technologies) following manufacturer’s instructions. Dye-labeled CD8⁺ T cells were then cultured on the anti-CD3/anti-CD28–coated plate at a density of 10⁵ cells per well in coculture media, and 2 μg/mL anti–IL-2 (using a mixture of both S4B6-1 and JES6-1A12 clones, Bio X Cell) was added at the beginning of coculture where indicated. The cells were cultured at 37°C and 5% CO₂ for 3 days. Following coculture, cells were stained and analyzed by flow cytometry.

An untreated, flat-bottom, 96-well plate was coated overnight at 4°C with 0.2 μg/mL anti-CD3 (clone 145-2C11, BD Biosciences) and 0.5 μg/mL anti-CD28 (clone 37.51, BD Biosciences) in PBS, washed with PBS, and blocked with coculture media (RPMI from Gibco containing 10% FBS from Atlanta Biologicals, 1% penicillin/streptomycin from Gibco, and 1× β-mercaptoethanol from Gibco) for at least 30 minutes at room temperature (RT). CD8⁺ T cells were isolated from the spleens of naive C57BL/6 mice using the untouched CD8⁺ T cell isolation kit (Miltenyi Biotec), following manufacturer’s instructions. Isolated CD8⁺ T cells were washed twice with PBS and stained with CTV dye (Life Technologies) following manufacturer’s instructions. Dye-labeled CD8⁺ T cells were then cultured on the anti-CD3/anti-CD28–coated plate at a density of 10⁵ cells per well in coculture media, and 2 μg/mL anti–IL-2 (using a mixture of both S4B6-1 and JES6-1A12 clones, Bio X Cell) was added at the beginning of coculture where indicated. The cells were cultured at 37°C and 5% CO₂ for 3 days. Following coculture, cells were stained and analyzed by flow cytometry.

For K562 cell apoptosis assay, purified human primary NK cells were initially activated with DMSO or Daphnetin for 18 h in 96-well V-plate. The K562 cells were labeled with CTV dye (ThermoFisher Scientific, Waltham, United States) and then added to the pre-activated NK cells for co-culture at a 5:1 ratio for another 6 h. The cells were harvested and then the apoptosis kit was used and the ratio of Annexin V^-7-AAD^-, Annexin V⁺7-AAD^-, and Annexin V⁺7-AAD⁺ K562 cells was determined by flow cytometry (18 (link)).
NK cell cytotoxicity assay against K562 cells was also performed by using a real-time digital bio-imaging system, as described previously (19 (link)). Briefly, purified human primary NK cells were initially activated with DMSO or Daphnetin in the presence of IL-12 for 18 h in a 96-well U plate. The K562 cells were labeled with CellTracker dye (ThermoFisher Scientific, Waltham, United States) and then added to the pre-activated NK cells for co-culture at different ratios. Next, the plate was placed in a cell-imaging multimode reader (Cytation 5, Biotek, VT, United States) and the protocol was set to focus on CellTracker-labeled K562 cells in each well at the indicated time points. The fluorescent area of CellTracker-labeled K562 cells in each well was recorded at 4 and 8 h and further analyzed using Gen5™ software (BioTek, VT, United States).