Relationships of diverse apoptotic death process patterns to mitochondrial membrane potential (Δψm) evaluated by three-parameter flow cytometric analysis

Abstract

Recently, it has been proposed that novel methodologies are needed to re-evaluate apoptotic cell death, as studies of apoptosis have shown it to be a complex process. Since mitochondria are key regulators in cell death pathways, we developed a simultaneous 3-parameter flow cytometric analysis that incorporates the change in mitochondrial membrane potential (Δψm) in an Annexin-V [for phosphatidyl-serine (PS)] and propidium iodide (PI) assay system (3 parameters with 4 colours), and evaluated the apoptotic process using various haematological malignant cell lines and death triggers. The present method enabled visualization of cell composition during apoptosis and captured complicated molecular events. For example, apoptotic cells that lost Δψm did not always externalize PS, while some late apoptotic cells had polarized Δψm. The findings of unchanged PS-externalization and aberrant cell death suggest that there is no relationship of PS externalization and apoptosis with an unknown apoptotic mechanism. Based on PS-externalization, sensitivity to staurosporine, and the combination of cell lines and triggers, the apoptotic process was classified into 2 types. Importantly, most of our findings could not be observed by PS–PI and Δψm assays when independently performed. Our method may be useful for examining mitochondrial-related apoptosis and death signalling pathways, as well as screening novel apoptosis-inducing cancer drugs.

Protective Effects of Salidroside on Mitochondrial Functions against Exertional Heat Stroke-Induced Organ Damage in the Rat

“…Mitochondrial membrane potential (ΔΨm) was detected using a fluorescent probe, tetramethylrhodamine methyl ester (TMRM; Molecular Probes Inc., Leiden, Netherlands), which reversibly accumulates according to the membrane potential. Briefly, isolated hepatocytes and cardiomyocytes were incubated in HEPES buffer (134 mmol/L NaCl, 6 mmol/L KCl, 2 mmol/L CaCl 2 , 1 mmol/L MgCl 2 , 10 mmol/L HEPES, and 10 mmol/L glucose; pH 7.40) containing TMRM (100 nmol/L) for 30 min followed by a 15-minute wash. Flow cytometric analysis (BD FACSCalibur) was performed to analyze mean fluorescence intensity (MFI) [ 11 ].…”

Optimization of PhysicoChemical Parameters for Production of Cytotoxic Secondary Metabolites and Apoptosis Induction Activities in the Culture Extract of a Marine Algal–Derived Endophytic Fungus Aspergillus sp.

“…To further evaluate the mechanism of ASE-mediated cell death in HeLa cells, phosphatidylserine (PS) externalization was measured using FITC-labeled annexin V (Koopman et al, 1994;Martin et al, 1995). PS externalization on the plasma membrane in apoptotic cells is an intermediate event, occurring after mitochondrial membrane depolarization but before DNA fragmentation during cellular apoptosis (Suzuki et al, 2013). Annexin-V FITC/propidium iodide-based flow cytometry was used to detect healthy cells, early, late apoptotic cells, or dead cells.…”

Laminaria japonica cyclic peptides exert anti‐colorectal carcinoma effects through apoptosis induction in vitro and in vivo

“…The mitochondria‐dependent pathway is the most common apoptotic pathway in vertebrate tumor cells, and Δψm collapse is one of the pivotal events of the apoptotic process. As previously reported, once oxidative stress reaches a certain level, Δψm is changed, resulting in the release of apoptosis factors [24, 25]. Furthermore, mitochondrial dysfunction, as indicated by the dissipation of Δψm, could subsequently cause the release of Cyt c from mitochondria into the cytosol [26].…”

“…The mitochondria-dependent pathway is the most common apoptotic pathway in vertebrate tumor cells, and Δψm collapse is one of the pivotal events of the apoptotic process. As previously reported, once oxidative stress reaches a certain level, Δψm is changed, resulting in the release of apoptosis factors [24,25]. Furthermore, mitochondrial F I G U R E 6 Effect of peritumoral injection of LCP-3 on the tumor size and weight mice were subcutaneously injected with CT26 cells (10 6 cells/mouse) into the right side of the back skin near the armpit.…”