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 pro cedures represent the only protection. Many influences of mycoplasma on cel l culture parameters have been described. We addressed the question of whet her mycoplasma contamination affects the most frequently used cytotoxicity assay, the tetrazolium based MTT assay. We contaminated C6 glioma cells wit h mycoplasma and performed MTT assays with doxorubicin, vincristine, etopos ide and cisplatinum under various conditions. Contaminated cells demonstrat ed significant different results when tested with the MTT assay than mycopl asma free controls. Differences were not detectable when cells were counted as toxicity assay. Due to an additional reduction of tetrazolium by mycopl asmas, contaminated cells appeared up to 15 fold resistant to doxorubicin, vincristine and etoposide, but not to cisplatinum. Differences decreased wi th decreasing drug doses and decreasing plated cell count. Our findings con firm the compelling need for periodical mycoplasma screening, especially wh en tetrazolium based cytotoxicity assays (MTT) are used.