CytoTox 96® Non-Radioactive Cytotoxicity Assay From Promega

The Promega CytoTox 96® Non-Radioactive Cytotoxicity Assay is an alternative assay format to standard ⁵¹Cr release cytotoxicity assays. This assay takes advantage of the stable cytosolic enzyme lactate dehydrogenase (LDH) which is released upon cell lysis. LDH activity in the cell culture supernatant is determined with a coupled enzymatic reaction, resulting in the conversion of the tetrazolium salt INT to a red formazan product, the amount of which is proportional to the number of lysed cells. Released LDH catalyzes the reaction of NAD+ and lactate to pyruvate and NADH, then NADH plus INT, in the presence of diaphorose, is catalyzed to NAD+ and formazan. Generation of Formazan is monitored by measuring absorbance at 490 nm.

The number of cells to be used and the background absorbance correction need to be addressed before performing this assay. As different cell types contain different concentrations of LDH, it is necessary to determine the optimum number of cells to use in the assay to in order to obtain a suitable signal to noise ratio. This optimization can be achieved by measuring LDH activity in serial dilutions of your cell type. Cell dilutions are prepared in a V-bottom 96-well plate and the cells are lysed using the provided lysis solution. The lysates are then transferred to another 96-well plate and the substrate mix is added. The lysate and substrate are mixed and incubated for 30 minutes at which point the stop solution is added and the absorbance at 490 nm measured. With this information, it is possible to determine the cell number which gives absorbance values at least two times the background absorbance of the medium. The background absorbance is the other consideration as there are a number of factors within the medium which can affect the absorbance (e.g. phenol red in the medium, LDH in animal sera). We have overcome the former by using phenol red-free medium and in some instances; we pre-incubate the cells in serum-free medium overnight before performing the assay. However, it is always necessary to run a medium alone sample to control for background absorbance due to the medium. We have used this assay successfully with a number of different cell lines including COLO205, A549, HeLa, and MCF-7.

This assay can be utilized to determine the cytotoxicity caused by chemical compounds, or cell-mediated cytotoxicity. We have used this assay principally to measure cell cytotoxicty caused by inhibitors of protein molecules involved in cell cycle control. The procedure for this type of assay is very simple. Once an optimal number of cells per well has been determined, this number of cells are plated in 96-well plates. Once plated, the cells are treated with the inhibitor, usually in serial dilutions, in duplicate or triplicate and allowed to incubate further. This incubation can be 24-72 hours in length depending on the mechanism of action of the inhibitor. To determine the maximum LDH activity, control wells with cells and vehicle alone are completely lysed with the provided lysis solution. Once the incubation and control lysis are completed, the 96-well plates are spun and the supernatants collected and transferred to another 96-well assay plate. Control wells with medium alone are also included to determine the background LDH activity.

At this point, the substrate mix is reconstituted and added to each well and incubated for 30 minutes at room temperature in the dark. At this juncture, the stop solution is added and the absorbance at 490 nm recorded. Background readings are subtracted from the sample readings and the % cytotoxicity is calculated using the sample LDH release (OD 490 nm) and the maximum LDH release (OD 490 nm) determined from the lysed cells.

In conclusion, with minimal optimization, the CytoTox 96® Non-Radioactive Cytotoxicity Assay represents a simple, reliable and robust alternative to traditional methods for determination of cytotoxicity.