An Introduction to Cytotoxicity Assays
Cytotoxicity assays are used in a variety of important medical research and industrial applications. In its simplest form, cytotoxicity refers to the ability of an agent (cellular, non-cellular) to cause a toxic effect (e.g., death or damage) to a target cell. Methods used to assess cell death or cytotoxicity have generally focused on three main biological aspects:
- Uptake of dyes such as trypan blue: cells that are dead are unable to perform the active transport of exogenous dyes
- Release of artificial labels (e.g., 51Cr, 111In)
- Release of endogenous molecules (e.g., LDH, GAPDH, DNA fragments)
Historically, the gold standard for measuring cell cytotoxicity has been the 51Cr-release method that detects the release of 51Cr from the cytoplasm of pre-labeled target cells after exposure to effector cells or other cytotoxic agents. This method relies on the agentís or effector cellís ability to initiate cell death, either by permeabilization of the target cell membrane or activating a biochemical pathway that results in cell death and disintegration. Cytotoxic T lymphocytes (CTL), natural killer (NK) cells, and lymphokine-activated killer (LAK) cells are all common examples of effector cells in cell-mediated cytotoxic models. However, "leaky" cells contribute to high spontaneous release of 51Cr from control cells, which diminish the signal-to-noise ratio. Other confounding factors include target cell damage caused by the radioisotope, which increases background and limits the experiment to the observation of cytotoxic events that must take place within 2-6 hours.
Current use of non-radioactive assay kits has reflected a trend in biomedical laboratories to avoid radioisotopes. Non-isotopic cytotoxicity assays include the LDH, G6PDH, or Adenylate Kinase (AK) enzyme release detection assays, and the ATP release detection assay, which monitor the release of cellular proteins or ATP from dying cells upon cell membrane disruption. While these methods have been useful in the past, they may severely underestimate the appearance of the cytotoxicity, because significant and relevant cell toxicity often occurs before cellular membrane permeabilization. That is, to cause cell membrane permeability, crucial intracellular pathways must be committed first, and then permeablization of the membrane occurs over time.
When studying cell-mediated or non-cell mediated (e.g., chemical, ADCC) cytotoxic effects, these types of enzyme or ATP based assays often suffer from large non-specific contributions from effector cells and non-treated control target cells (i.e., the spontaneous release of the endogenous protein or ATP from the effector cells, and the target cells). This non-specific signal results in a relatively low dynamic range for the assay and causes the assay to be generally less sensitive in detecting the appearance of cytotoxic effects associated with the target cells. In the case of LDH, it is known that serum-derived LDH adds to the background and the LDH test may have to be performed at a lower serum concentration than is optimal for the cells in the assay. In addition, the quality and viability of the assayed cells must be very high in order to reduce the spontaneous release of the LDH, G6PDH, ADH enzyme or ATP from the control target cells as much as possible.
Other non-isotopic cytotoxicity assays measure cell proliferation and death indirectly by detecting and quantitating the increase or decrease of the cellular metabolic activity after exposure to cytotoxic agents. These types of assays include ones such as MTT, XTT or WST-1 colorimetric assays that detect formazan products from mitochondrial metabolism. A major shortcoming to these indirect methods has been high background signals that are due to the spontaneous release of metabolic markers from all viable cells. This non-specific signal results in a relatively low dynamic range for the assay and causes the assay to be significantly less sensitive in detecting the appearance of cytotoxic agent-induced cytotoxic effects associated with the treated target cells. In addition, these assays are dependent on metabolic processes that may not detect early events of cell death. It is also critical for the background signal to be as low as possible in order to detect the effect of cytotoxicity on target cells. Again the quality and viability of the assayed cells must be very high in order to minimize the spontaneous assay signal from the target cells as much as possible.
The assays discussed above are not optimally suitable for detecting the death of small numbers of target cells in a population of many cells because these assays indirectly measure the activity of all viable cells present.
In contrast, the Ziva®-Tox assay directly measures cytotoxicity based on the measurement of a) the extent of BrdU label incorporation into cytotoxic agent treated target cells during or after cytotoxic agent treatment, relative to the extent of DNA synthesis in non-treated control target cells (i.e., measures the extent of cellular DNA synthesis inhibition caused by the treatment with the cytotoxic agent) or b) the extent of loss of BrdU labeled target cell DNA from cytotoxic agent treated target cells (i.e., target cells which are pre-labeled with BrdU and then treated with the cytotoxic agent), relative to the extent of loss of pre-labeled BrdU DNA from the non-treated target cell control.