Optimized alamarBlue assay protocol for drug dose-response determination of 3D tumor spheroids

The assessment of drug-dose responses is vital for the prediction of unwanted toxicological effects in modern medicine. Three-dimensional (3D) cell cultures techniques can provide in vivo-like spheroids and microtissues that resemble natural tumor function. However, formation of necrotic core and diffusion limitation of chemical compounds within these models can reduce the reproducibility and precision of standard bioassay protocols used to test two-dimensional (2D) cell cultures. Nonetheless, the accurate prediction of detrimental effects of test compounds based on functional bioassays is essential for the development of new efficient therapeutic strategies. For instance, alamarBlue(®) is a widely-used commercially available redox indicator dye that can evaluate metabolic activity and cellular health status in a single-step procedure however, suitability and optimization of this bioassay must be determined for each individual application scenario. Here, we optimized the standard alamarBlue(®) proliferation/viability protocol for tumor spheroid cultures to enhance assay precision during toxicological drug screening. We optimized the original protocol of alamarBlue(®) assay that usually suggests an incubation time of 2–4 hours. The key modifications of the protocol for spheroid cultures are as follows: • Aspiration of cell culture medium before drug exposure. • Replacement of drug-supplemented medium with 10% (v/v) alamarBlue(®) reagent mixed with culture medium. • Increase of incubation period to 24 h at 37 °C protected from light.