Cells were seeded in 96-well plates at a density of 1.25×10 4 cells per well for 24 hours and subjected to treatments. Cellular viability was assessed using the MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (Thermo Fisher Scientific, Waltham, Massachusetts). For colony formation assay, cells treated with defined conditions were further cultured for 10 to 12 days. Colonies were stained with 0.5% crystal violet solution and counted using ImageJ software (NIH, Bethesda, Maryland). Flow cytometrybased cell death assessment was performed using annexin V-FITC/propidium iodide (PI) staining (Thermo Fisher Scientific) and were analyzed using FlowJo software (FlowJo LLC, Ashland, Oregon). Data of combined drug effects were analyzed by the Chou and Talalay method using CompuSyn software (13) . Combination index values of less than 1, 1, and more than 1 indicated synergism, additive effect, and antagonism, respectively.
Annexin 5 fitc propidium iodide staining
Manufactured by Thermo Fisher Scientific
Annexin V-FITC/propidium iodide (PI) staining is a fluorescent dye-based assay used to detect and quantify apoptosis (programmed cell death) in cells. Annexin V binds to phosphatidylserine, which is externalized on the cell surface during apoptosis, while PI stains the nucleic acids of cells with compromised cell membranes. This combination of dyes allows for the discrimination between viable, early apoptotic, and late apoptotic/necrotic cells.
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2 protocols using annexin 5 fitc propidium iodide staining
1
Cytotoxicity Assessment of Drug Combinations
2
Cytotoxicity Assessment of Drug Combinations
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Cells were seeded in 96-well plates at a density of 1.25×10 4 cells per well for 24 hours and subjected to treatments. Cellular viability was assessed using the MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (Thermo Fisher Scientific, Waltham, Massachusetts). For colony formation assay, cells treated with defined conditions were further cultured for 10 to 12 days. Colonies were stained with 0.5% crystal violet solution and counted using ImageJ software (NIH, Bethesda, Maryland). Flow cytometrybased cell death assessment was performed using annexin V-FITC/propidium iodide (PI) staining (Thermo Fisher Scientific) and were analyzed using FlowJo software (FlowJo LLC, Ashland, Oregon). Data of combined drug effects were analyzed by the Chou and Talalay method using CompuSyn software (13) . Combination index values of less than 1, 1, and more than 1 indicated synergism, additive effect, and antagonism, respectively.
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