Cell death is neither a binary phenomenon nor an instantaneous event. Before a cell dies-whether from a physiological or pathological stimulus-complex, carefully orchestrated molecular pathways are activated that determine whether a cell survives, adapts or succumbs. Understanding these processes prior to the point of no return is fundamental in multiple fields: from cell and molecular biology to pharmacology, to oncology and regenerative medicine.
For decades, cell death was classified in a binary manner: apoptosis (programmed, orderly death) or necrosis (disordered, traumatic death). However, today we know that there are multiple types of programmed or regulated cell death, each with distinct biochemical, morphological and functional characteristics.
It is a fundamental process for embryonic development, tissue homeostasis and the elimination of damaged or dangerous cells. It involves sequential activation of caspases, loss of mitochondrial potential, exposure of phosphatidylserine (PS) at the plasma membrane and DNA fragmentation.
There are two main pathways:
-Intrinsic (mitochondrial) pathway: regulated by the Bcl-2 family and activated by intracellular stress such as ADn damage or oxidative stress.
-Extrinsic pathway (death receptors): induced by ligands such as FasL, TNF-α or TRAIL.
A form of morphologically regulated cell death similar to necrosis, but dependent on specific proteins such as RIPK1, RIPK3 and MLKL. It is relevant in inflammatory diseases, viral infections and cancer.
More and more specific pathways are being identified, posing new challenges for their detection and therapeutic modulation.
One of the major advances in biotechnology has been the development of tools to detect early events in the cell death cascade, before irreversible morphological signs appear. This allows early intervention, understanding molecular mechanisms and predicting cellular response to drugs or toxic agents.
Phosphatidylserine, normally confined to the inner face of the plasma membrane, is exposed at the cell surface during the initial stages of apoptosis. This event is detected with high sensitivity by annexin V conjugated to fluorochromes (such as FITC or APC), usually combined with propidium iodide (PI) to distinguish viable, early apoptotic and necrotic cells.
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Mitochondrial dysfunction is an early event in the intrinsic apoptosis pathway. It is measured by lipophilic probes such as JC-1, TMRE or DiOC6, which change their fluorescence as a function of mitochondrial energy status.
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Caspases are key proteases in the execution of apoptosis. There are fluorogenic or colorimetric kits based on specific substrates such as DEVD-AFC for caspase-3 or IETD-AFC for caspase-8.
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Increased ROS can be both a trigger and a consequence of cell death. The DCFDA (or H “DCFDA) probe allows quantification of intracellular ROS by fluorescence.
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Probes such as propidium iodide (PI) or 7-AAD penetrate only into cells with compromised membranes. They are useful to identify necrosis or late stages of apoptosis.
Combining these assays in multi-parametric formats, especially by flow cytometry or high-throughput automated platforms, allows:
These analyses not only save time and costs, but also improve safety and efficacy in the development of new treatments.
The study of events prior to cell death opens a critical window of time: the moment when intervention is possible. In contexts such as cancer, degenerative diseases or immune therapies, knowing when and how a cell is starting its path towards death allows us to design more effective therapeutic strategies, reduce side effects and personalise treatments.
Thanks to today’s tools, cell death is no longer a silent end; it is an opportunity to understand, prevent and transform.