The challenge to measure cell proliferation in two and three dimensions.

Various assays, using different strategies, are available for assessing cultured cell proliferation. These include measurement of metabolic activity (tetrazolium salts and alamarBlue), DNA quantification using fluorophores (Hoechst 33258 and PicoGreen), uptake of radioactively-labeled DNA precursors such as [3H]thymidine, and physical counting (hemocytometer). These assays are well established in characterizing cell proliferation in two-dimensional (2D), monolayer cultures of low cell densities. However, increasing interest in 3D cultures has prompted the need to evaluate the effectiveness of using these assays in high cell density or 3D cultures. We show here that typical cell proliferation assays do not necessarily correlate linearly with increasing cell densities or between 2D and 3D cultures, and are either not suitable or only rough approximations in quantifying actual cell numbers in a culture. Prudent choice of techniques and careful interpretation of data are therefore recommended when measuring cell proliferation in high cell density and 3D cultures.