Table 1.

Considerations in performing MTT assay.

Does the MTT assay represent what you aim to measure? Have you optimized the following parameters? ▪ cell seeding number and density ▪ MTT concentration ▪ MTT incubation time Do your culture conditions (such as culture media type, presence of serum, and phenol red in the media, etc.) affect your assay measurement by either optical or chemical interference? Are any effects of tested treatments considered that could affect the final OD measurements in a direct or indirect way? ▪ MTT uptake and/or extrusion (e.g., cell membrane permeability/integrity) ▪ Cell number (e.g., proliferation) ▪ Cell metabolism (e.g., chemo/radio-induced senescence-like phenotype) ▪ Cell secretome (e.g., chemo/radio-induced senescence-associated secretory phenotype) ▪ Background absorbance and scattering ▪ Abiotic reduction of MTT

If your aim is a quantitative measurement of cell viability, have you defined how OD values relate to the number of cells?

Based on your answers to the above questions: ▪ Is MTT assay an appropriate tool to answer your research question? ▪ Have you considered using appropriate control samples to reduce risk of misinterpretation of data? ▪ Have you considered complementary assays to confirm MTT assay results?