Igg1κ isotype control

Target cells were incubated with effector NK92 cells at an E/T ratio 1:1 for 4 h in the presence of anti-CD107a antibodies or IgG1κ isotype control (BD Biosciences). For the last 3 h, cells were treated with BD Golgi Stop (BD Biosciences). After washing, cells were stained with anti-CD56 antibodies (BioLegend) or IgG1κ isotype control (BD Biosciences). Fluorescence was immediately measured with a BD LSRFortessa X-20. The results were analyzed with FlowJo software. The all experiments were performed at least twice, and the representative results are presented as mean ± SD.

HT-29 cells were seeded in 60-mm dishes and treated at 50% confluency according to indicated treatment conditions. Hydrogen peroxide (2 mM H₂O₂) was used as positive control and was added to the cells for 30 min (22 h 30 min after hypericin activation), and afterwards, the medium was replaced with fresh medium for 1 h. Cells were then harvested by trypsinization at 24 h after hypericin activation, fixed, permeabilized, and washed using BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (BD Biosciences), according to the manufacturer’s instructions. After 20 min of incubation in 3% FBS and centrifugation, cells were stained for 1 h at room temperature in the dark with antibodies against either IgG1 κ Isotype Control (1:10, BD Biosciences) or histone H2AX phosphorylation on Ser139 (γH2AX) (1:10, BD Biosciences), which is a marker of DNA double-strand breaks (DSBs). Staining intensity was quantified by flow cytometry (BD FACSCalibur) and FlowJo software (TreeStar Inc.). The expression of γH2AX was expressed as a ratio of the median fluorescence of anti-H2AX (pS139) to that of IgG1 κ Isotype Control.