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 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.

Synthetic flavagline derivative 1‐chloroacetylrocaglaol promotes apoptosis in K562 erythroleukemia cells through miR‐17‐92 cluster genes

“…Previous works have illustrated that the advancement of apoptosis in tumor cells by flavaglines is the essential mechanism underlying the inhibition of tumor cell growth. [28,29] Similarly, in the current work, A2074 contributed to cell-cycle arrest in K562 cells through apoptosis. Hoechst staining visualized apoptotic nuclei with condensed chromatins.…”

Hydrogen Sulfide-Evoked Intracellular Ca2+ Signals in Primary Cultures of Metastatic Colorectal Cancer Cells

“…( E ), mean ± SE of ΔΨ m in the absence (Ctrl) and presence of NaHS (100 µM, 72 h). ΔΨ m was measured by evaluating tetramethylrhodamine, methyl ester (TMRM) fluorescence [ 67 ].…”

Increased oxidative stress and mitochondrial impairments associated with increased expression of TNF‐α and caspase‐3 in palmitic acid‐induced lipotoxicity in myoblasts

“…Using the microscopy method, cells labeled with DAPI has been shown to identify apoptosis based on nuclear morphology. [ 63,64 ] As represented in Figure 4 and Figure 5A–C our results showed that the palmitic acid treatments (50–150 μM) for 24 and 48 h enhance the apoptosis in the L6 myoblasts. Similar studies were conducted in the human vascular endothelial cells, showing apoptosis triggered by the saturated fatty acid.…”