Investigating the health of cells is a foundational aspect of biological research and medicine. Examining cell health helps scientists understand basic biological processes, disease progression, and treatment effects. It also allows researchers to assess the impact of experimental conditions, ensuring reliable results. This provides insight into normal cell function and what goes wrong in disease, guiding new therapies.
Understanding Cell Damage
Cytotoxicity refers to the property of a substance causing damage or death to cells. Damage can include loss of membrane integrity, halted growth, or programmed cell death. Cytotoxic agents, such as certain chemicals, drugs, or environmental pollutants, can disrupt cellular processes, leading to these detrimental effects.
Lactate Dehydrogenase (LDH) is a stable enzyme located within the cytoplasm of nearly all living cells. Under healthy conditions, LDH remains confined inside the cell. However, when the cell membrane is damaged due to injury or cell death, LDH leaks into the extracellular environment (e.g., cell culture medium). The amount of released LDH indicates cell membrane integrity loss and cell damage.
How the Assay Works
The LDH cytotoxicity assay quantifies the amount of LDH released from damaged cells into the cell culture medium. The underlying principle involves an enzymatic reaction catalyzed by the leaked LDH. Specifically, LDH converts lactate into pyruvate, reducing nicotinamide adenine dinucleotide (NAD+) to NADH.
The newly formed NADH then participates in a second coupled reaction, reducing a tetrazolium salt into a colored product called formazan. The intensity of this color is directly proportional to the amount of LDH in the sample, correlating with the number of damaged or lysed cells. This color change is measured using a spectrophotometer, usually at 490 nm or 500 nm.
The general steps involve culturing cells and treating them with the substance or condition being investigated. After incubation, the supernatant is collected. Specific assay reagents are then added to this supernatant, initiating the enzymatic color-producing reaction. Finally, the color intensity is measured using a spectrophotometer to determine the level of cell damage.
Applications and Practical Considerations
The LDH cytotoxicity assay is a widely used tool in various scientific fields due to its straightforward nature and reliable results. It screens potential drug candidates for toxic effects on cells. The assay also assesses the toxicity of environmental pollutants on biological systems, providing data for safety evaluations.
In cancer research, the LDH assay evaluates anti-cancer therapies by measuring cancer cell death in response to treatment. It also serves as a general method for studying cell health, offering insights into cellular responses to various stimuli. The assay is compatible with both adherent cells and cells in suspension, making it versatile for different experimental setups.
Practical considerations are important for accurate interpretation of LDH assay results. While the assay is simple, fast, and non-radioactive, it primarily measures cell membrane integrity loss. It does not distinguish between different mechanisms of cell death.
The presence of serum in cell culture media can also contribute to background signals, as serum contains some LDH activity. To mitigate this, researchers use minimal serum concentrations or include appropriate controls. Proper controls are crucial for calculating accurate cytotoxicity percentages.