Cellular toxicity is an essential component of most culture-based research experiments. The impact of pharmaceuticals, chemicals, viruses, bacteria, other cells and other environmental factors on cell viability is often one of the primary outcome measures or a principle pilot study of most research. Lactate dehydrogenase, the cytosolic enzyme that catalyzes the transformation of pyruvate to lactate, is released from the cell upon loss of plasma membrane integrity or necrosis. The CytoTox 96 Non-Radioactive Cytotoxicity Assay from Promega is an easy-to-use, 2 step, colorimetric kit that provides a non-radioactive method for measuring this LDH release in the culture media.
The kit comes with a substrate mix that is stored as a lypholized powder and provided in 4 vials for easy reconstitution and use. In addition, the kit contains an assay buffer to reconstitute the substrate mix and a stop buffer to complete the reaction. Once reconstituted, the substrate mix can be stored at -20oC for 6 – 8 weeks and we have found that there is no loss in effectiveness if stored at 4oC and protected from light for 1 – 2 weeks. The final elements of the kit are a lysis solution and a positive control to ensure that the kit is working. The assay is simple and involves the addition of 50 ul of culture media to 50 ul of substrate mix, a 30 minute incubation in the dark, addition of the stop buffer and analysis of colorimetric changes. The addition of the reaction buffer results in a conversion of a tetrazolium salt into a red formazan product that can be measured with a 96-well plate reader. Enough supplies are provided in the kit for 1000 assays. At $213/kit, this works out to approximately $20 per 96-well plate, which makes the kit relatively expensive, but considering the ease of use, this can be considered a fair trade.
In addition to its simplicity, the kit offers a good deal of flexibility. For example, it can be used to determine the percent cell death of a specific cell type, by assessing the maximal cell death (using the provided lysis buffer) and the amount of LDH release after intervention. In addition, because the kit measures the amount of LDH released into a 50 ul aliquot of culture media, it is possible to measure cell lysis over time by repeating the media extractions. It is also possible to remove the media for the LDH assay and retain the cells for immunocytochemistry, cell viability assessment with the CellTiter 96 Non-Radioactive Cell Proliferation Assay, Trypan Blue confirmation or any number of other measurements.
We have been using the CytoTox 96 kit for several years and I have found the kit to be an incredibly simple, easy to use and reliable method for detecting cell death in a number of culture systems. I have used the kit to detect cell death in both primary neuronal and microglial cultures, after both cell-mediated and pharmaceutical interventions, and found it easy to customize and adjust to both systems. The alternative method in use in the lab is the standard Trypan Blue exclusion method, which is, in comparison, subjective and far less straight forward than the CytoTox 96 Assay.
In summary, we have found the CytoTox 96 Assay to be a valuable laboratory tool that is now used routinely by all of the laboratory members in a number of different culture systems for a number of different interventions. The method is easy, reliable, fast and flexible for a number of different investigations.
Kimberly Byrnes
Postdoctoral Fellow
Department of Neuroscience
Georgetown University