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.

According to the World Health Organization (WHO), cancer is one of main causes of death worldwide, with 8.2 million people dying from this disease in 2012. Because of this, new forms of treatments or improvement of current treatments are crucial. In this regard, Photodynamic therapy (PDT) has been used to successfully treat cancers that can be easily accessed externally or by fibre-optic endoscopes, such as skin, bladder and esophagus cancers. In addition, this therapy can used alongside radiotherapy and chemotherapy in order to kill cancer cells. The main problem in implementing PDT is penetration of visible light deeper than 10 mm in tissues, due to scattering and absorption by tissue chromophores. Unfortunately, this excludes several internal organs affected by cancer. Another issue in this regard is the use of a selective cancer cell-photosensitizing compound. Nevertheless, several groups have recently developed scintillation nanoparticles, which can be stimulated by X-rays, thereby making this a possible solution for light production in deeper tissues. Alternative approaches have also been developed, such as photosensitizer structure modifications and cell membrane permeabilizing agents. In this context, certain channels lead to transitory plasma membrane permeability changes, such as pannexin, connexin hemmichannels, TRPV1-4 and P2×7, which allow for the non-selective passage of molecules up to 1,000 Da. Herein, we discuss the particular case of the P2×7 receptor-associated pore as a drug delivery system for hydrophilic substances to be applied in PDT, which could also be carried out with other channels. Methylene blue (MB) is a low cost dye used as a prototype photosensitizer, approved for clinical use in several other clinical conditions, as well as photodynamic therapy for fungi infections.

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