## 3 Results
### Viability Assay and Data Normalization  
To investigate MB dark toxicity and phototoxicity on the SHSYSY, U87, and GL261 cell lines, those cells were incubated with 1 to 400 uM MB without light for 30 min at 37 \({}^{\circ}\)C and 5% CO\({}_{2}\). Half of them were then exposed to red light. Given inter-experiment variability, the literature describes a variety of normalization methods to allow for comparison among results (Table 4). We compared these different methodologies, both those used with protocols for MTT assays [27] as well as some that were proposed for HTS assays [48; 49; 50]. Normalization methods, in general, produce high-quality response curves that allow a reliable characterization of cytotoxic effects while mitigating false positive or false negative test results; the chosen methodology was the one that best met this purpose. The normalization methods evaluated were separated into two sets: control-based normalization methods--CBN (Figure 4A-C) and non-control-based normalization methods--NCBN (Figure 4D-F). All CBN methods showed the same differences between treatments, with a clear difference between positive (no treatment) and negative (0.4% Triton-X) controls. The NCBN methods (Figure 4D-F) do not require a positive or negative control to compare treatments as they use data of all the wells as an internal control; the samples are considered ineffective or inactive. Subsequently, these samples could be used as controls [51]. In the case of the Z-score, a D'Agostino-Pearson normality test was performed. It is important to mention that, in these types of analyses, we wanted to detect the outliers, data points not consistent with all other measurements [50]. Commonly, Z-scores with an absolute value of > 3 or < -3 are considered outliers from a normal distribution. The choice of 3 as the threshold for the absolute value (k = 3) originates the 3-sigma rule\" [52]. \begin{table}
\begin{tabular}{p{113.8 pt} p{113.8 pt} p{113.8 pt}} \hline \hline
**Method** & **Equation** & **Observations** & **Reference** \\ \hline _Controls-based_ & & & \\ \hline Percent of control & \(POC\ =\ \frac{X_{l}}{Z}\times 100\%\) & & where _Xi_ is the raw measurement of treatment and \(\hat{\mathbb{C}}\) is the mean of the measurements of the positive controls & [43; 49] \\ \hline \% Maximum data Absorbance & \(\%MAb\ =\ \frac{x_{l}}{MAb}\) & & where _Xi_ is the raw measurement of treatment and _MAb_ is the maximum data absorbance of assay & W. R. \\ \hline Percent of Median of control & \(POMC\ =\ \frac{x_{l}}{M\ of\ control}\) & & where _Xi_ is the raw measurement of treatment and _“M of control”_ is the median of the measurements of the positive controls & [50] \\ \hline _Non-controls-based_ & & & \\ \hline \(Z\) score & \(Z\ =\ \frac{(X_{l}-\hat{\mathbb{C}})}{S_{x}}\) & & where _Xi_ is the raw measurement on the compound, \(\hat{\mathbb{C}}\) and _Sx_ are the mean and the standard deviation, respectively, of all measurements within the plate & [51] \\ \hline Percent of samples & \(Z\ =\ \frac{X_{l}}{\hat{\mathbb{C}}}\times 100\%\) & & where _Xi_ is the raw measurement on the compound and \(\hat{\mathbb{C}}\) is the mean of all measurements within the plate & [50] \\ \hline Robust Percent of samples & \(Z\ =\ \frac{X_{l}}{Med}\times 100\%\) & & where _Xi_ is the raw measurement of the compound and _Med_ is the median of all measurements within the plate & [50] \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Normalization methods. As seen in Figure 4, concentrations ranging from 5 to 100 \(\upmu\)M show a similar distribution, with their z-scores between 0 and 1. However, a clear difference was observed for the concentration of 400 \(\upmu\)M (z-score = \(-\)2). Although the 3 sigma rule cannot be applied in this particular case, it is considered that at this concentration there is a significant effect on cell viability after photodynamic treatment. Therefore, statistical and graphical methods can be used to identify the outliers, allowing the identification of those photosensizers with significant activity.