Falsification of tetrazolium dye (MTT) based cytotoxicity assay results due to mycoplasma contamination of cell cultures

Mycoplasma contamination of cell cultures is a frequently observed problem. Due to the inconspicuous growth in cell cultures, periodical screening procedures represent the only protection. Many influences of mycoplasma on cell culture parameters have been described. We addressed the question of whether mycoplasma contamination affects the most frequently used cytotoxicity assay, the tetrazolium based MTT assay. We contaminated C6 glioma cells with mycoplasma and performed MTT assays with doxorubicin, vincristine, etoposide and cisplatinum under various conditions. Contaminated cells demonstrated significant different results when tested with the MTT assay than mycoplasma free controls. Differences were not detectable when cells were counted as toxicity assay. Due to an additional reduction of tetrazolium by mycoplasmas, contaminated cells appeared up to 15 fold resistant to doxorubicin, vincristine and etoposide, but not to cisplatinum. Differences decreased with decreasing drug doses and decreasing plated cell count. Our findings confirm the compelling need for periodical mycoplasma screening, especially when tetrazolium based cytotoxicity assays (MTT) are used.