###### 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-dimethylthiazol2-y)l-2,5-diphenyltetrazolzolium 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. MIT assay; cell MTT metabolic activity; photodynamic therapy +
Footnote †: journal:co-enzyme production, cell adherence, ATP production, and nucleotide uptake activity (Table 1) [21]. The most suitable assay depends on the type and quantity of cells, on the mechanism of cell death induced by the photosensitizer [24; 25], and also on the cost and complexity of each test. Due to its low cost and ease of implementation, the MTT assay [26] has been extensively employed over the past 30 years to evaluate new PDTs, and its use continues to increase: Between 2006 and 2019 the number of publications found in Google Scholar using \"MTT assay\" and \"Photodynamic Therapy\" as search terms increased by a factor of 10, reaching more than 2000 in 2019 (Figure 1). Nonetheless, some important technical complexities and limitations can lead to inappropriate use of the MTT assay. For instance,  
\begin{table}
\begin{tabular}{p{12.3 pt} p{12.3 pt} p{12.3 pt} p{12.3 pt} p{12.3 pt}} \hline \hline
**Name** & **Methodological Mechanism’s Summary** & **Detection Method** & **Advantage** & **Disadvantage** & **Ref. ** \\ \hline MTT Assay & MTT (2-(4,5-dimethyl-2-thiazol))-3,5-diphenyl-2H-tetrazol bromide), a yellow tetrazole, is reduced to purple formazan in living and metabolic active cells through an enzymatic reaction & Spectrophotometer/ & -Fast protocol; -High & -Overestimation of viability; -Final solubilization step; -Reducing compounds are identified to interfere with tetrazolium reduction assays & [26; 27] \\ MTT Assay & Activated respiring cells convert the XTT (sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenyl)-carbonyl]-2H-tetrazolium inner salt) to a water-soluble, orange colored formazan product & & & \\ WST-1 Assay & & & & \\ WST-1 Assay & & & & \\ WST-1 occurs primarily at the cell surface & & & & \\ MTS (5-[3-(carboxy)]) & & & & \\ (5-[3-(carboxy)])-3-(4-nitrophenyl-2-thiazol))-2-(4-sulfo-phenyl)-2H-tetrazolium inner salt) in the presence of phenazine methosulfate & Spectrophotometer/ & -Highest sensitivity; -Faster protocol & -Endpoint assay; -Overestimation of viability & [29] \\ MTS Assay & & & & \\ MTS Assay & & & & \\ WST-1 assay & & & & \\ WST-1 occurs primarily at the cell surface & & & & \\ MTS (5-[3-(carboxy)]) & & & & \\ WST-10, phenyl]-3-(4,5-dimethyl-2-thiazol)-2-(4-sulfo-phenyl)-2H-tetrazolium inner salt) in the presence of phenazine methosulfate & & & & \\ WST-10, PDMS, produces a formazan product & & & & \\ WST-10, PDMS, & & & & \\ WST-1  
excess MTT salt concentration can lead to nonspecific cell death depending on the cell type [32] and cell density directly correlates with resistance to oxidative stress in vitro [33].