RRC ID 54083
著者 De P, Carlson JH, Leyland-Jones B, Williams C, Dey N.
タイトル Triple Fluorescence staining to Evaluate Mechanism-based Apoptosis following Chemotherapeutic and Targeted Anti-cancer Drugs in Live Tumor Cells.
ジャーナル Sci Rep
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 in A2780 and OVK18 cells following drug treatment which decreased proliferation, & increased apoptotic signaling with mitochondrial depolarization. Treatment blocked cytoplasmic MitoViewBlue staining while increased both nuclear NucView488-Casp3 substrate and red membranous CF594 AnnexinV staining. Merged images showed 100% mutual exclusivity between MitoViewBlue and caspase3 or AnnexinV stains in control and treated cells as determined by overlap and colocalization coefficients. Caspase3 and AnnexinV staining in treated cells were both separate and overlapped (yellow fluorescence) indicating the sequence of apoptotic-events. The protocol will help in deciphering mechanistic involvement of different stages/features of apoptosis in tumor cell following anti-cancer drugs in real-time.
巻・号 8(1)
ページ 13192
公開日 2018-9-4
DOI 10.1038/s41598-018-31575-3
PII 10.1038/s41598-018-31575-3
PMID 30181562
PMC PMC6123436
MeSH Antineoplastic Agents / pharmacology* Apoptosis / drug effects* Cell Line, Tumor Drug Screening Assays, Antitumor / methods Female Fluorescence Humans Microscopy, Fluorescence / methods* Mitochondria / drug effects Mitochondria / pathology Ovarian Neoplasms / drug therapy Ovarian Neoplasms / pathology Signal Transduction / drug effects Staining and Labeling / methods
IF 3.998
引用数 1
リソース情報
ヒト・動物細胞 OVK18(RCB1903)