## 3 Results  
### Cell Density for the MTT Reduction Assay in PDT  
A key question when using a viability test is whether cell density could influence the assay, as cells at lower density are more affected by the photosensitizer (PS) treatment [43]. Therefore, it is necessary to establish the appropriate number of cells/well to be used. In this study, this was tested taking, as a starting point, data from the literature reporting cell densities ranging from 2 \(\times\) 10\({}^{3}\) to 4 \(\times\) 10\({}^{4}\) for U87, 1 \(\times\) 10\({}^{3}\) to 4 \(\times\) 10\({}^{4}\) for SHSYSY, and 4 \(\times\) 10\({}^{3}\) to 2 \(\times\) 10\({}^{4}\) for GL261 cell line.  
Figure 2 shows the effect of increasing cell densities on OD\({}_{570}\) in an MTT protocol with or without incubation with Triton X-100 (0.4%). As cell density increased, so did the difference between treatments, in an S-shaped curve (insets, Figure 2A-C). For the SHSYSY (Figure 2A) and U87 (Figure 2B) cell lines there was no difference between treatments (\(p\) > 0.001) at the lowest densities (1 \(\times\) 10\({}^{3}\) and 2 \(\times\) 10\({}^{3}\), respectively), whereas for the GL261 cell line, Triton yielded a significantly higher absorbance at 4 \(\times\) 10\({}^{3}\) cells/well. The effect of cell density saturated at 3 \(\times\) 10\({}^{4}\), 2 \(\times\) 10\({}^{4}\), and 1.2 \(\times\) 10\({}^{4}\) cells/well, for the SHSYSY, U87, and GL261 cell lines, respectively (Figure 2A-C).  
Calculating the Z'factor for each cell line, we found that densities of 1-3 \(\times\) 10\({}^{4}\) cells/well yielded the values closest to 1 h for all cell lines (Table 3). As a result, these densities were used in the PDT assays.  
\begin{table}
\begin{tabular}{c c} \hline
**Z-Factor** & **Interpretation** \\ \hline
1.0 & Ideal \\ Between 0.5 and 1.0 & Excellent assay \\ Between 0 and 0.5 & Marginal assay \\ \hline \end{tabular}
\end{table}
Table 2: Interpretation of the calculation of the Z’factor.  
Figure 2: Standardization of the cell inoculum for the MTT reduction assay. (**A**) SHSYSY, (B) U87, and (**C**) GL261 cell lines. The graphics show the effect of increasing cell densities on OD\({}_{570}\) in an MTT protocol with or without incubation with Triton X-100 (0.4%). As cell density increased, so did the difference between treatments, in an S-shaped curve (**A**–**C**). For the SHSYSY (**A**) and U87 (**B**) cell lines, there was no difference between treatments (\(p>0.001\)) at the lowest densities (\(1\times 10^{3}\) and \(2\times 10^{3}\), respectively), whereas for the GL261 cell line, Triton yielded a significantly higher absorbance at \(4\times 10^{3}\) cells/well. The effect of cell density was saturated for the SHSYSY, U87, and GL261 cell lines at \(3\times 10^{4}\), \(2\times 10^{4}\), and \(1.2\times 10^{4}\) cells/well, respectively (**A**–**C**). Data are expressed as mean \(\pm\) standard deviation. To test the homogeneity of variance, we used [PERSON]’s test. For the statistical comparison of the data, we used two-way ANOVA followed by Sidak’s post-test (\(p<0.001\)), ns = not significant, (\({}^{*}=p<0.05\); **** = \(p<0.001\)). Graphs represent three independent experiments performed in triplicate.