A modified methylene blue assay for accurate cell counting

Cell counting is a common technique in cellular and molecular biology research applications, such as cell culture maintenance, cell plating, cell growth and cell doubling time determinations, as well as cell proliferation and cytotoxicity measurements. Many commonly employed cell counting methods exhibit limitations that influence resulting accuracy or versatility. For example, the trypan blue method typically underestimates cell numbers in culture, and the Lowry protein assay can be influenced by cell cycle. An urgent need exists for a method of cell counting that is both accurate and versatile. This work intended to explore an adaptation of the methylene blue assay to overcome the existing limitations of the procedure, enabling application to a broader range of cell densities and various cell culture plates. This new methylene blue assay was found to be more efficient, accurate and sensitive. A linear relationship (r2 > 0.99) was established between cell number and absorbance at 570 nm wavelength when the new methylene blue assay was applied to three cell lines (HepG2, Caco-2, and MCF-7) plated in a broad range of cell densities (5 × 104 to 2.5 × 106) in four different types of culture plates (6-, 12-, 24-, and 96-well plates). Growth curves were determined using both the trypan blue and methylene blue methods. At each time point in the HepG2 growth curve, the cell count obtained using the trypan blue assay was statistically significantly lower than that obtained using the methylene blue assay (p < 0.05). The same was true for the Caco-2 growth curve at all time points (p < 0.05) except at the 0 h. The methylene blue method proposed in this paper may serve as a direct, automated counting method for cells grown in any type of culture plates. This assay has clear advantages over traditional methods and is a powerful tool for any application requiring a versatile, efficient, and accurate method of cell counting, such as bioavailability and cytotoxicity assays, and more basic experiments such as cell growth curve or doubling time determination, especially in the research of natural products, bioactive compounds, phytochemicals, functional foods and nutraceuticals.