Our initial experiments included a direct comparison of ATP assay reagent from two commercial sources. To visualize the lytic process, we combined a DNA binding dye (CellTox™ Green Cytotoxicity Assay) with the ATP detection reagent. The DNA binding dye is not permeable to live cells, but becomes fluorescent when it binds to DNA in cells that have lost membrane integrity. We could view spheroids treated with that combination of reagents to see which cells became stained after membrane disruption. As expected, we observed a substantial difference in the lytic capacity of different commercial reagents and, even though the CellTiter-Glo® Assay was superior, the results suggested the need to further characterize and improve assays applied to 3D culture models.
Additional experimental work led to development of a new reagent formulation (CellTiter-Glo 3D Assay) that more effectively lyses cells in large 3D spheroids and extracts ATP. The new formulation contains a higher detergent concentration, which was enabled by the properties of a recombinant form of luciferase that is stable to harsh reagent conditions. The protocol incorporates better mixing and longer exposure to the lytic reagent.
The Table illustrates a comparison of the recovery of ATP from different sizes of 3D spheroids using different reagents. Acid extraction of samples and comparison to ATP standards was used to measure the efficiency of ATP extraction. We also have confirmed improved performance of the new CellTiter-Glo 3D Assay with other models that use hydrogel and inert scaffolds.