Triple Fluorescence staining to Evaluate Mechanism-based Apoptosis following Chemotherapeutic and Targeted Anti-cancer Drugs in Live Tumor Cells.

ABSTRACT: We present a protocol for live cancer cell-imaging by triple-fluorescent staining to test 3 crucial mechanisms of apoptosis; the enzymatic activity of executioner caspase3, caspase-dependent phosphatidylserine presentation on the cell surface and mitochondrial function. We standardized a protocol to co-stain live tumor cells with the NucView488-Casp3 substrate, CF594 AnnexinV, and MitoViewBlue. We validated this protocol following apoptosis induction with paclitaxel or in combination with BKM120. Fluorescent imaging of cells using simultaneous live/dead cell markers (CalceinAM green/EthD-1red) was used as internal control. We used quantitative confluence (Essen), AnnexinV-PE staining (Accuri C6), expression of cl-caspase3, Cl-PARP and mitochondrial potential (TMRE-A) as validation criteria

SUBMITTER: De P 

PROVIDER: S-EPMC6123436 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature