Neutral Red Uptake Assay (NRU-Test)


The NRU-Test can be used to test for cytotoxic effects of chemical substances and environmental samples on cell membranes. It is based on a dyeing reaction which allows identification of dead and live cells. Usually the test is performed with the adherent permanent cell lines HEP-G2 (human hepatocyte cells) and V79 (lung epithelium of the Chinese hamster). In addition, several other cell lines like human keratinocytes or fish cells can also be used.


In the microwell plate version, a monolayer cell culture, which has been grown over a period of 24 hours, is exposed over a defined period of time (usually between 2 and 24 hours) to different concentrations of the test substance. By adding a rat liver homogenate (so-called S9-mix) the metabolic activation of toxic substances in the organism can be simulated. At the end of the exposure period, the cells are washed and the neutral red dye is added. It passes the cell membrane and binds to intracellular phosphate and carboxyl groups. The accumulation of the dye takes place mainly in the lysosome. Dead cells or such with membrane damage cannot accumulate the dye, so that during the following washing and fixation steps the dye is not retained intracellularly. Wells with dead or damaged cells or with a reduced number of cells due to growth inhibitive effects are less coloured than those with vital cells. The evaluation of the test is done by resolving the intracellular bound dye and measuring the intensity photometrically in comparison to an untreated control sample. The toxicity is expressed as percent inhibition of neutral red dye retention in the sample. It is a measure for cell damage. EC50 can be calculated from the dose-response curve.