## 4 Discussion  
This article aimed to identify key factors to be taken into account when designing in vitro PDT assays employing MTT as a cell viability test. Cell density has been shown to modify the sensitivity of the cells to toxins, environmental stress, and extrinsic apoptotic signaling. For example, the cytotoxicity of ascorbic acid for malignant lymphoid CEM-C7 cells significantly increases when cell density decreases [53]. [PERSON], et al. [33], demonstrated that cell density directly correlates with resistance to oxidative stress in vitro. On the other hand, if cells get to the stationary phase, the OD\({}_{570}\) is not linear [47, 54]. In our study, the OD\({}_{570}\) values decreased at the highest cell density, probably because cells had stopped dividing as a result of cell accumulation, shape change, and growth factor depletion. These results indicated that U87, SHSY5Y, and GL261 cells should be seeded at a density lower than 2 \(\times\) 10\({}^{4}\)/well and suggested that experiments using longer periods should be designed in such a way as to avoid an over confluence that could lead to depletion of nutrients from the culture medium and, therefore, lower cell viability. If the PS to be evaluated presents long-term toxicity, it is advisable to choose viability assays other than MTT [55]. Figure 5: Dose-response curves of Methylene Blue. Dose-response curves in (**A** and **B**) SHSY5Y human neuroblastoma, (**C** and **D**) U87 glioblastoma, and (**E** and **F**) GL261 cell lines. The concentration of MB produced a 50% response of the maximal response (EC\({}_{50}\)) calculated in absence of light, and after 6 min exposure at 18–23 mW/cm\({}^{2}\). Data are expressed as mean \(\pm\) SEM. The data were normalized as a function of the mean negative control treatment (CN = cells without MB). The Z\({}^{\prime}\)-factor values for \(1\times 10^{4}\) to \(2\times 10^{4}\) cells/well were 0.8 and 0.9, respectively, which should provide a greater working range (positive and negative controls can be discriminated from each other), using the Z'factor calculations [52]. The advantage of using the Z'factor is the reduction of the amplitude and variability of the assay signal to only one parameter [56]. Several publications have described the use of Z'factor as a measure of quality control. For instance, [PERSON] et al. used Z'factor to demonstrate the utility of the SAMDI approach for chemical screening against protease activity of lethal anthrax factor [57]. In the same line, [PERSON] et al., compared the performance of Alamar blue and MTT cell viability assays in a high throughput format, reporting Z\({}^{\prime}\)-factor values of 0.85 and 0.82 for the Alamar blue and MTT assays, respectively. These values indicate that both assays are of high quality [58]. Regarding the conditions of the MTT assay, using the concentration described in the literature [42; 59; 60] we observed data fluctuation in some cases. MTT reduction can vary significantly between cell lines [61], and the formazan that results from MTT reduction can exhibit harmful effects, such as cell membrane damage during its exocytosis, inducing cell death [62]. [PERSON] et al. [27], described morphological changes in Balb/c 3T3 cells during incubation with MTT solution at 0.8 mg/mL. Thus, we evaluated the effect of several MTT concentrations on the survival of the U87 cell line. Our results are consistent with a cytotoxic effect of MTT concentrations higher than 0.500 mg/mL (Figure 3). In general, it is accepted that the MTT assay measures a set of enzymatic activities, which are related to cellular metabolism in different ways. The first description of MTT reduction by the mitochondrial electron transport chain was in the 1960s; since then, the mitochondrial II/succinate dehydrogenase complex has been erroneously seen as the unique site of intracellular reduction of MTT [63]. However, there is evidence of non-mitochondrial MTT reduction, through oxidoreductaseuates, which use NADH/NADPH as cofactors.