Considerations and Technical Pitfalls in the Employment of the MTT Assay to Evaluate Photosensitizers for Photodynamic Therapy

Abstract

Photodynamic therapy (PDT) combines light, a photosensitizing chemical substance, and molecular oxygen to elicit cell death and is employed in the treatment of a variety of diseases, including cancer. The development of PDT treatment strategies requires in vitro assays to develop new photosensitizers. One such assay is the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide developed in 1983 and widely used in PDT studies. Despite the exponential growth in the number of publications, a uniform MTT protocol for use in the PDT area is lacking. Herein, we list and standardize the conditions to evaluate the photosensitizer methylene blue (MB) in glioblastoma and neuroblastoma cell lines. In addition, we review technical pitfalls and identify several variables that must be taken into consideration in order to provide accurate results with MTT. We conclude that for each cell line we must have a dose-response curve using the MTT assay and good controls for the standardization. Additionally, the optimal values of the time and cell density must be in the linear range of the curve to avoid errors. We describe all relevant points and outline the best normalization techniques to observe the differences between treatments.

The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis

“…However, increasing MTT concentration from 0.4 to 0.5 mg/mL did not result in any further increase in OD levels, regardless of cell number and measurement time point ( Figure 6 ). A decrease [ 16 ] or plateau [ 18 ] in OD levels above a certain MTT concentration was also reported previously and shown to vary depending on the tested cell type. A likely explanation for these observations is an acceleration of MTT-induced cell death at higher MTT concentrations.…”

“…Reaching a plateau level in OD beyond a certain number of cells has also been reported previously [ 16 ], attributed to the over confluence caused by seeding high numbers of cells and consequent changes in nutrient availability and cell viability and metabolism [ 16 , 22 ]. The fact that we did not observe a saturation level in OD by increasing cell number, is not an unexpected finding given the maximum seeding number used in this work (20,000 cells/well of Corning ® 96-well Microplate, area per well: 0.32 cm 2 ) is expected to correspond to approximately 90% confluence at the time of the measurements (27–30 h after seeding) based on our light microscopy observations.…”

“…As the data indicates, regardless of MTT concentration and MTT incubation time, increasing cell seeding number increases OD ( Figure 2 ). This is intuitive as increasing the number of cells accordingly increases the total level of produced formazan by the cell population and consequently the measured OD, as shown in other studies [ 16 , 17 , 18 ]. The fact that the OD levels in the absence of MTT did not change with increasing the cell number shows this is not due to the optical effects of increased cell number ( Figure 3 and Figure 4 a).…”

“…However, at the lower levels of cell number and/or MTT concentration, the maximum (saturation) level of MTT reduction is not reached up to 4 h of MTT incubation. In other words, the saturation time point depends on the seeded cell number and MTT concentration, as previously shown [ 16 ].…”

“…This also provides a direct indication that changes in the OD levels are not necessarily a direct measurement of changes in the percentage of viable cells, i.e., doubling the cell number does not result in a doubling of OD level. Other studies have also shown that OD is not always a linear function of cell number, and the function varies depending on cell type [ 16 , 18 ], cell number [ 16 , 17 , 18 ], pH [ 18 ], and the formazan-solubilizing agent [ 17 ]. Moreover, it was previously shown that a higher number of cells could cause a shift in the UV-Vis spectrum peak possibly due to changes in pH.…”