Bodipy 665 676

Cells were treated as indicated; then, 50 μM BODIPY™ 665/676 (Thermo Fisher Scientific, USA) was added to the cells, which were incubated for 1 h. Excess BODIPY™ 665/676 was removed by washing the cells twice with PBS. Representative images were obtained using a confocal microscope.

4T1 cells were seeded in confocal cell culture dishes at a density of 1 × 10⁴ cells per dish. After culture in incubator for 12 h, HSN (final concentration: [pTBCB] = 50 µg mL⁻¹), HSN with DEVD (100 µM), HSN with DFO (100 µM), and PBS were, respectively, added to the cells. After incubation for 24 h, cells were gently washed three times with fresh PBS and then fixed with 4% paraformaldehyde. After fixation, cells were incubated with PBS containing 0.1% Triton X-100 (PBST) and then washed three times with ice-cold PBS. Afterwards, cells were incubated with 3% bovine serum albumin (BSA) in PBST for 30 min. Then cells were washed three times in PBS and incubated with primary cleaved caspase-3 antibody (Cell Signaling Technology) (1:1000 in PBST) in a humidified chamber at 4 °C overnight. After incubation, cells were washed three times in PBS and incubated with secondary antibody Alexa Fluor 488 conjugated donkey anti-rabbit IgG (Thermo Fisher Scientific) (1:1000 in PBST) at room temperature for 1 h. Then cells were washed with PBS and sequentially stained with BODIPY 665/676 (Thermo Fisher Scientific) (10 µM, 30 min) and DAPI. After mounted by Fluoromount aqueous mounting medium, cells were imaged under a LSM800 confocal microscope.

The lipophilic and oxidation-sensitive dye BODIPY 665/676 was purchased from Thermo Fischer (Waltham, MA, USA). l-ascorbic acid, sodium chloride (>99.5%, EMSURE^®), and alumina power (Alumina N—Super I) were obtained from Aldrich-Europe (Darmstadt, Germany), MilliporeSigma (Burlington, MA, USA), and MP EcoChrom™ (Eschwege, Germany), respectively. Spirit white vinegar (4%), soybean oil, and egg yolk containing 8% (w/w) NaCl were purchased from a local store. Demineralized (demi) water was used for all experiments.