The neutral red assay can be used to evaluate cell viability during autophagy or in an acidic microenvironment in...

Harsh conditions within the tumor microenvironment, such as hypoxia and extracellular acidic pH (pH_e), inactivate some chemotherapies, which results in limited or no cytotoxicity. Standard MTT, ATPlite and protease assays that are used to determine the potency of newly developed drugs often give erroneous results when applied under hypoxic or acidic conditions. Therefore, development of a cytotoxicity assay that does not yield false positive or false negative results under circumstances of both hypoxia and acidic pH_e is needed. We evaluated currently used cell viability assays as well as neutral red staining to assess viability of ovarian and pancreatic cancer cells grown in an acidic pH_e microenvironment after treatment with carboplatin, gemcitabine or chloroquine. We validated cell viability using western blotting of pro-caspase-9 and cleaved-caspase-9, and LC3-I and - II. Standard cell viability assays indicated cell viability accurately at pH_e 7.4, but was not correlated with induction of apoptosis or autophagy at acidic pH_e. By contrast, our modified neutral red assay detected cell viability accurately over a range of pH_e as demonstrated by its correlation with induction of apoptosis and autophagy. Neutral red staining is effective for evaluating the effect of chemotherapeutic agents on cell viability under acidic pH_e or hypoxic conditions.