Quantifying Cellular Apoptosis by Flow Cytometry

The cellular apoptosis was evaluated following the protocol as previously described by our group (Rosenberger et al., 2019 (link)). Briefly, 6,250 cells/cm² were seeded in 100 mm plates (Falcon, Franklin Lakes, NJ, United States, Cat. #353003) in maintenance medium. After 48 h, the culture medium was removed, and the cells were washed three times with phosphate-buffered saline (PBS 1×) before starting the culture in the different induction medium: (a) DMEM high glucose + 1% L-glutamine; (b) Oxium^TMEXO (Consorcio Regenero S.A., Las Condes, Santiago, Chile; patent No. PCT/CL2019/100175, Tapia-Limonchi et al., 2019 ); or (c) commercial medium (RoosterBio Inc., Frederick, MD, United States, Cat. #M2001). After 6 days, cell supernatants were mixed with the trypsinized cells in order to include detached dead cells in the analysis. Then, cells were stained with Annexin V-APC (BioLegend, San Diego, CA, United States, Cat. #640920) and 7-aminoactinomycin D (7-AAD) (BioLegend, San Diego, CA, United States, Cat. #420403) in Annexin V binding buffer (BioLegend, San Diego, CA, United States, Cat. #422201). The analysis was performed by flow cytometry using a FACSCanto^TM II cytometer (BD Biosciences, San Jose, CA, United States). The data acquired were analyzed using the FlowJo software V10 (Tree Star, Ashland, OR, United States).