Quantitative Analysis of Apoptosis and Necrosis in Live Cells Using Flow Cytometry.

Apoptosis and necrosis are the two sides of the cell death penumbra. Apoptosis is a well-studied model of cell death wherein the cell destroys itself employing a predefined form of active signaling without the release of soluble cytoplasmic contents to the external environment. Compared to apoptosis, necrosis is a nonspecific form of sudden cell death in response to an invasive external stimulus which in turn is devoid of active programmed intracellular signaling leading to the sudden release of the soluble cellular contents consequent to the rupture of the cell membrane. This fundamental difference between apoptosis and necrosis made us believe that the former is the safe form of cell death and the latter is an undesirable one which often elicits an inflammatory response to the adjacent cells. Recent studies have shown that necrosis also involves a few defined cellular and complex biochemical events similar to apoptosis rendering it difficult to distinguish these two events at the single-cell level using the currently used popular assays.Here we provide a newly described detailed methodology encompassing cell system development along with a multiparametric flow cytometry-based approach to discriminate apoptotic cells from necrotic cells using a stable cell line expressing genetically encoded probe for detecting caspase activation and DsRed targeted at the mitochondria.

Mitochondria targeted redox GFP reveals time and dose dependent onset and progression of mitochondrial oxidation with diverging cell death decisions during photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) is a successful cancer treatment modality. In vitro, in vivo, and clinical studies with different photosensitizers reveal diverging cell fates, including apoptosis, necrosis, autophagy, and non-specific forms of cell death. The mode of action and efficacy of PDT is mediated through free radical generation and is highly dependent on diverse variables such as nature, dose, metabolism of photosensitizer, irradiation energy, and irradiation cycle. AIM: Discovery of newer photosensitizers and optimization of PDT approaches to achieve a clinically relevant form of cell death called apoptosis requires better in vitro real-time methods. Oxidative damage and mitochondrial permeabilization are critical signaling events involved in photodamage and apoptosis. Hence, mitochondrial damage detection is an appropriate target signaling for mechanistic evaluation of PDT. METHODOLOGY: We report mitochondria-targeted redox GFP expressing cells as a sensitive system to test and validate important variables of PDT using the photosensitizer 5-Aminolevulinic acid (5-ALA) as a model. An independent FRET-based caspase sensor cell was also used to study the impact of the photosensitizer dosage and irradiation duration on the mode of cell death. RESULTS: The study reveals that the cancer cells expressing mt-roGFP are extremely sensitive to monitor mitochondrial oxidation induced by PDT. The extent of mitochondrial redox changes induced by PDT can be determined using these sensor cells by real-time image-based approaches. These approaches provide sufficient temporal resolution that is required to fine-tune and optimize the PDT conditions. The degree of oxidation of the probe is highly dependent on the dosage of photosensitizer and duration of light irradiation, which determines the nature of cell death. A real-time caspase sensor probe further confirmed that the caspase-dependent and caspase-independent nature of cell death is in high correlation with the extent of mitochondrial oxidation. A condition that triggers rapid and extreme mito-oxidation seems to favor necrosis, while delayed and slowly progressing redox changes contribute to caspase-dependent apoptosis. CONCLUSION: The study confirms that temporal analysis of mitochondrial oxidation is a reliable biomarker for fine-tuning PDT conditions to achieve the desired outcome. This can be achieved using stable cancer cell lines expressing mitochondria-targeted roGFP by ratiometric imaging.