Cellmask blue

Cells grown in 96-well plates were fixed and permeabilized with a 2% formaldehyde solution in methanol, for 20 min at − 20 °C. Quenching of formaldehyde was achieved by incubation for 15 min with a 0.1 M glycine solution and blocked with a 1% FBS solution for 30 min. Cells were then incubated for 1 h at room temperature with primary antibodies rabbit anti-Caspase-8 IgG mAb (1:400, cleaved caspase-8, Cell Signaling Technology, Cat. No. 9496) or rabbit-anti-Caspase-9 IgG mAb (1:400, cleaved caspase-9, Cell Signaling Technology, Cat. No. 9502), followed by incubation for 45 min with a solution containing secondary antibody donkey anti-Rabbit DyLight 488 (1:300, ThermoFisher, Cat. No. 35553), as well as nuclear and cytoplasmic dyes Hoechst or CellMask Blue (2.5 μg/mL, ThermoFisher, Cat. No. H32720).

Cells were plated on coverslips and fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA). Cells were permeabilized using 0.5% Triton-X in PBS and blocked using 10% bovine calf serum in PBS, followed by immunostaining. Immunofluorescence images were obtained using a Nikon Eclipse 80i fluorescence microscope using NIS elements software to deconvolve and focus stacked images or the Operetta™ high content imager (PerkinElmer, Waltham, MA) as indicated.
For Operetta™ imaging, cells were plated in 96-well cell-carrier plates (PerkinElmer) and imaged in a single focal plane at ×20 magnification. Using Columbus™ (PerkinElmer) software, the protein intensity in the cytoplasm and nucleus were quantified. The nucleus and cytoplasm were defined by DAPI staining and CellMask™ Blue (Thermo Fisher Scientific) staining, respectively. The percentage of protein in the nucleus was calculated as the ratio of signal intensity present in the nucleus divided by the signal intensity present in the entire cell. The percentage of protein present in the nucleus was calculated for every cell and median values reported per well. To compare treatment conditions, the average of these median values was calculated for multiple wells within each independent experiment.

Cells were seeded in a 384-well plate and incubated with either complete medium or in the presence of 250 μM H₂O₂ at 37°C for 2 hr. Cells were then pulsed with Alexa-647 Tfn (10 µg/ml) for 5 min, followed by 3x PBS wash, fixed with 3.7% PFA for 15 min, and then stained with DAPI (1:1000) and CellMask Blue (1:2000) (ThermoFisher). Image acquisition was performed via the automated confocal imaging system, CV7000S Yogokawa. Images analysis were performed using MotionTracking software.