Fluoview ix81 confocal microscope

HeLa cells were purchased from ATCC® (#CCL-2). HeLa cells (seeding population: 8.0 × 10⁴ cells/well) were incubated in EMEM (+10% FBS) in 6-well glass bottom plates at 37 °C. At 70–80% confluence, media was aspirated, and cells were washed twice with PBS and serum-free EMEM was added to wells. After incubation for 16 h at 37 °C the media was aspirated, and cells were washed twice with PBS. For GOX treatment, cells were treated with CysOx probe and GOX at the indicated concentrations in PBS containing 0.1% DMSO and glucose (1 mg/mL). At the indicated time points, wells were analyzed using an Olympus FluoView IX81 confocal microscope using FV10-ASW software v3.0. For tBOOH treatment, oxidant was added at the indicated concentrations in EMEM for 10 min at 37 °C followed by the addition of CysOx probe at the indicated concentration for additional 15 min at 37 °C. The media was then aspirated, cells washed twice with PBS, fresh PBS was added, and wells were analyzed as above. Fluorogenic CysOx signal was visualized using the 458 nm laser channel, filtered using a SDM560 dichroic mirror, followed by BA505-605 band pass filter. Five frames were recorded per condition. Images were analyzed using ImageJ software v1.52a, where the pixel intensity of the cellular cytoplasmic regions was measured with at least 10 measurements per condition.

HeLa cells (8.0 × 10⁴ cells/well) were incubated in EMEM (+10% FBS, supplemented with penicillin/streptomycin) in six-well plates with a glass bottom for imaging (Chemglass, Cat. No. CLS-1812-006). Culture media was aspirated, cells were washed twice with PBS, EMEM lacking FBS was added to the wells, and cells were incubated at 37 °C for 16 h. Culture media was aspirated, and cells were washed twice with PBS. Wells were treated with CysOx1 or CysOx 2 (50 µM final concentration) and an organelle-selective dye (Invitrogen, ER-tracker red, Cat. No. E34250 or Cell Signaling Technology, Mitotracker Deep Red FM, Cat. No. 8778 S) in EMEM with 0.1% DMSO for 1 h at 37 °C. Selected wells were also treated with H₂O₂ (300 µM final concentration). After incubation, culture media was aspirated, cells were washed twice with PBS, fresh PBS was added, and analyzed using an Olympus FluoView IX81 confocal microscope using FV10-ASW software v3.0.