Apoptosis wash buffer

Live cell staining for high-content image analysis: cells were added to a CellCarrier-96 Ultra Microplates (Perkin Elmer, Waltham, MA, USA), 1000 cells/well. Fam/Cas dye (1:150) was added and incubated for 1 h and washed with apoptosis wash buffer (Life Technologies V35118; diluted 1:10). Hoechst 1:200, ToPro-3 1:1000, and TMRM 1:100,000 (Invitrogen, Waltham, MA, USA) were added and incubated for 10 min, then replaced with medium (without indicators) and analysed by an Operetta platform (Perkin Elmer). Results were interpreted using the Columbus™ software (Perkin Elmer). Annexin staining was carried out with an Annexin V FITC labelling kit (Miltenyi Biotec), analysed by a Cytoflex S flow cytometer (Beckman Coulter, Indianapolis, IN, USA) using a 488/525 nm filter.

Live cell staining for high content image analysis: cells were added to a CellCarrier-96 Ultra Microplates (Perkin Elmer), 1000 cells/well. Fam/Cas dye (1:150) was added and incubated for 1 h and washed with apoptosis wash buffer (Life Technologies V35118; diluted 1:10). Hoechst 1:200, ToPro-3 1:1000 and TMRM 1:100,000 (Invitrogen) added and incubated for 10 min, then replaced with medium (without indicators) and analysed in an Operetta platform (Perkin Elmer). Results were interpreted using the Columbus™ software (Perkin Elmer).
To assess if autophagy acts in SM-induced cell death, acridine orange (ImmunoChemistry Technology) was used to show if SM increased acidic properties within the cell (acidic organelles, AOs), indicating increased lysosome content. CPCs were plated in 12-well plates and allowed to reach confluency, then 5 µM acridine orange was added to medium for 30 min. Cells were PBS-washed once and imaged using confocal microscopy (Zeiss LSM880). The number of positive cells was quantified using ImageJ cell counter function (National Institutes of Health).