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Reactive oxygen species (ROS) are chemically active, oxygen-derived molecules with unpaired electrons that make them unstable. They form primarily in mitochondria when electrons leak during cell metabolism and the electron transport chain. The addition of one unpaired electron to stable dioxygen creates the superoxide anion, the initial ROS form, which then generates other reactive species like hydrogen peroxide and hydroxyl radicals.

Oxidative stress occurs when ROS levels exceed the cell's antioxidant defense capacity, creating a positive feedback loop of damage. These highly reactive molecules oxidatively damage cell membranes, DNA, and proteins. Excessive ROS has been implicated in cancer initiation and progression, and induces cellular senescence, supporting the Free Radical Theory of Aging that links ROS to cellular aging and death.

Beyond their harmful effects, ROS serve important physiological functions. During immune responses, phagocytes generate excessive ROS in a respiratory burst to oxidatively degrade pathogens. ROS also act as necessary intermediates and regulators in various cell signaling pathways and can signal the death of cancerous cells, making them essential mediators of cellular defense and communication.

H2DCFDA is a non-fluorescent probe that passively diffuses into cells. Intracellular esterases cleave its acetate groups, making it polar and retained within the cell. Upon oxidation by ROS, the hydrogen atoms are removed, converting non-fluorescent H2DCFDA into highly fluorescent dichloro-fluorescein (DCF), which can be quantified using plate readers, flow cytometers, or fluorescence microscopy.

The protocol begins by transferring cultured cells to phosphate buffered saline and centrifuging to wash them. Add H2DCFDA probe solution and incubate cells in the dark to prevent photobleaching. After incubation, wash cells to remove unloaded dye and transfer to a plate. Add oxidative stress inducers as needed, then analyze using a plate reader set to fluorescein excitation and emission wavelengths.

Researchers employ ROS detection to investigate phagocytosis and immune development. In one study, zebrafish treated with NADPH inducers showed higher ROS at 72 hours post-fertilization than at 48 hours, revealing developmental differences in immune response. Additionally, scientists measure ROS levels in neurons and astrocytes to identify mitochondrial dysfunction and assess how astrocytes protect central nervous system neurons from oxidative stress.

ROS levels reflect the balance between electron transport during cell metabolism and antioxidant defenses. Measuring ROS provides insight into cell metabolism metabolic pathways and assays, revealing how metabolic activity generates oxidative stress. Since mitochondrial dysfunction and excessive ROS are pathological features of many diseases including cancer and neurodegeneration, reliable ROS quantification is invaluable for disease research and understanding cellular health.