As with the classic CellTiter-Glo® Reagent, the CellTiter-Glo® 3D Reagent generates a luminescent signal that is proportional to the amount of ATP present using pure ATP (Figure 1, Panel A), and the amount of ATP is directly proportional to the number of cells present in 2D cell culture (Figure 1, Panel B).
Figure 1. Luminescence generated by the CellTiter-Glo® Assay is proportional to ATP and/or cell number. Panel A. Different concentrations of ATP in water were plated at 100 µl and assayed using the classic CellTiter-Glo® or CellTiter-Glo® 3D Reagents. Panel B. Dilutions of Jurkat cells were prepared in RPMI 1640 with 10% FBS and plated at 100 μl cells per well. An equivalent volume of CellTiter-Glo® 3D Reagent was added to all samples, and after 2 minutes of shaking, the luminescence was recorded at 10 minutes.
However, for microtissues in 3D culture, the correlation between cells seeded and luminescent signal is often not linear. For example, with hanging-drop spheroids produced using the InSphero GravityPLUS™ 3D Culture and Assay Platform (Figure 2, Panel A), effects such as contact inhibition on cell proliferation and reduced metabolic activity in the central region of large spheroids cause the relationship between seeded cell number and luminescent signal to be curvilinear (Figure 2, Panel B). However, by including CellTox™ Green Dye, a cell membrane-impermeant fluorogenic DNA-binding dye, in our lytic assay we can show that as microtissue size increases, the amount of ATP detected (luminescence, RLU) by the CellTiter-Glo® 3D Assay is proportional to the amount of DNA detected (fluorescence, RFU; Figure 2, Panel C). This confirms that the CellTiter-Glo® 3D Assay is effectively lysing cells, resulting in release of ATP, and can accurately report the number of viable cells in microtissues produced through 3D cell culture.
Figure 2. Seeding density vs. size, ATP content and DNA content. Panel A. Differential contrast images of HCT116 colon cancer spheroids grown for 4 days in the InSphero GravityPLUS™ 96-well hanging-drop platform after seeding with 400, 800, 1600, 2400, 3200, 4800, 6400, or 8000 cells. Images were acquired in an InSphero GravityTRAP™ plate. Panels B and C. An equivalent volume of CellTiter-Glo® 3D Reagent was added to all samples, and after 5 minutes of shaking, luminescence and/or fluorescence was recorded at 30 minutes. In Panel C, a 2X concentration of CellTox™ Green Dye was added to the CellTiter-Glo® 3D Reagent prior to sample addition.