## 4 Discussion
#### Viability Assay and Data Normalization  
Normalization of quantitative assay data is critical when interpreting effective biological system status to reduce fluctuations in the raw data between independent experiments and allow greater confidence in data comparisons [27; 31; 48]. Accordingly, we compared normalization methods (Table 4), which we divided into two categories: (1) controls-based (CB) methods, which, as the name suggests, assume that their controls are 100% or/ and 0% of some effect--these methodologies have a significant problem when the controls do not work properly; (2) non-controls based (NCB) methods, which do not use comparisons with controls, but rather comparisons between samples, relying on the general distribution of values rather than depending on controls [51]. Some groups are using NCB normalization with the median, not the mean because this parameter is not affected by outliers. The percent of control method is the most used in the MTT literature, and the other five normalization methods tested here provided similar results. Still, we suggest using at least one CB and one NCB test in MTT PDT assays to better visualize the data. Besides always making the experiments at least in triplicate will help evidence any outliers. Photodynamic therapy (PDT) is a procedure based on the utilization of light-activated compounds or photosensizers (PS). The rational discovery and design of PS molecules have been the subject of exhaustive research efforts of investigators [68]. Over the years, several techniques have been developed to screen candidate molecules, but most of these techniques are labor-intensive, time-consuming, produce waste products that are toxic or radioactive, and lastly, are expensive to perform [24; 64]. In contrast, the MTT assay is a simple, low-cost procedure that can be adapted well to PDT protocols. In addition, toxicity is observed with the MTT assay at lower concentrations compared to the assays required for detection of toxicity with the lactate dehydrogenase leakage, the neutral red, and Alamar blue assays [51; 65]. Ultimately, as mentioned, the MTT assay measures the metabolic activity of cells, including some mitochondrial and cytosolic dehydrogenases. Since apoptosis is a common response to PDT [27; 42], this can be a reasonable method for detection of efficacy, as it will likely reflect the numbers of functional mitochondria and therefore survival. However, if other death pathways are involved, it can be inaccurate. With that in mind, MTT assay is just a quick test for phototoxicity that needs to be supplemented with clonogenicity appraisal. Clonogenic assays are frequently used to study the survival of irradiated cancer cells [43], though MTT assays are widely accepted to study chemosensitivity or toxicity of drugs in human tumor cell lines. In the literature, specific studies on the comparability of MTT and clonogenic assays with positive results can be found [44; 45]. Equally relevant to consider is how the effects vary with the time between PDT treatment and carrying out the assay of MTT. For more precise results, the MTT test can be performed several hours (24 to 48 h after irradiation) after PDT treatment because you can have late repair or loss of toxicity. Nevertheless, it is important to consider in PS screening that a variety of chemical compounds might affect the MTT assay result due to their ability to lead to a non-enzymatic reduction of the MTT to formazan, such as dithiothreitol [34], vitamin A, and ascorbic acid [35]. Moreover, the MTT assay should not be used in the presence of mitochondrial uncoupling agents, both natural and synthetic [36; 69]. Plant extracts and polyphenic compounds also have been reported to interfere with the MTT assay [37]. The use of a control without cells in the MTT assay has been proposed to avoid false-positives resulting from the presence of molecules with reducing properties, such as flavonoids, but evidence in the literature suggests that this may not be enough [70].