Vybrant did dye

For NK cell-induced cell death, HeLa cells were seeded in 24-well plates overnight. Cells were loaded with 5 μM calcein AM (eBioscience) before co-incubating with NK-92 MI, YT or primary NK cells at E: T ratios = 3:1. 2 or 3 hours after incubation, calcein release was determined by recording fluorescence at 528 nM after excitation at 485 nM using a BioTek Synergy plate reader. For flow cytometry analysis, cells were collected by trypsinization and stained with annexin V conjugates (ThermoFisher A23204) and PI (Abcam). Samples were immediately analyzed by flow cytometry. For Vybrant DiD staining, HeLa cells were seeded overnight and stained with Vybrant DiD dye (ThermoFisher) according to the manufacturer’s instructions, and then NK-92 MI cells were added at an E: T ratio of 3:1. Pyroptotic cells were imaged using EVOS FL cell imaging system.

Raptinal, EGTA, necrostatin-1s (Nec-1s), ferrostatin-1 (Fer-1), α-tocopherol (Vit. E), and desferoxamine (DFO) were from Sigma. TRIzol was from Life Technologies. TRAIL was from Enzo Life Sciences. zVAD-fmk and zDEVD-fmk were from BD Biosciences. SYTOX green was from Invitrogen. Vybrant DiD dye was from ThermoFisher Scientific. CellTiter 96 kit (Promega) was used to measure cell proliferation.