## 2 Materials and Methods
### In Vitro Cell Viability  
To study the effects of microalgal extracts and Pheophorbide \(a\) (CAS15664-29-6; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) on cell viability, A2058, HepG2 and HACAT cell lines were seeded in 96-well microtiter plates (\(1\times 10^{4}\) cells-well\({}^{-1}\)) and incubated at 37 \({}^{\circ}\)C for 24 h. Then, fresh medium was used, including extracts and Ppa: (1) 50 ug\(\cdot\)mL  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Species** & **Abb** & **Class** & **Growth Media** & \
\begin{tabular}{c} **Sampling** \\ **Location** \\ \end{tabular} &
\begin{tabular}{c} **Concentration at** \\ **the End of the** \\ **Stationary Phase** \\ \end{tabular} \\ \hline _Asterionellosis glacialis_ & _Ag_ & Fragilariophyceaeae & F/2 & English Channel & \(10^{5}\) \\ _Chlamydomonas_ sp. & _CspA_ & Chlorophyceaeae & F/2-Si & Mediterranean Sea & \(10^{6}\) \\ _(CCMP2536) & _CspB_ & Chlorophyceaeae & F/2-Si & Nantucket Sound & \(10^{6}\) \\ _Cylindrotheca closterium_ & _Cc_ & Bacillariophyceaeae & F/2 & Mediterranean Sea & \(4\times 10^{5}\) \\ _Dunaliella tertiolecta_ & _Dt_ & Chlorophyceaeae & F/2-Si & Unknown & \(2\times 10^{6}\) \\ _Dityulum brightwellii_ & _Db_ & Mediophyceaeae & F/2 & Mediterranean Sea & \(10^{5}\) \\ _Trieres mobiliensis_ & _Om_ & Cosinchosophyceaeae & F/2 & Mediterranean Sea & \(10^{5}\) \\ _Pseudo-nitzchia arenysensis_ & _Pa_ & Bacillariophyceaeae & F/2 & Mediterranean Sea & \(3\times 10^{5}\) \\ _Pseudo-nitzchia fraudulenta_ & _Pf_ & Bacillariophyceaeae & F/2 & Mediterranean Sea & \(3\times 10^{5}\) \\ _Rhinomonas reticulata_ (CCAP & _Rr_ & Cryptophyceaeae & F/2 & Mediterranean Sea & \(10^{5}\) \\ _Scrippsiallacuminata_ & _St_ & Dinophyceaeae & K & Mediterranean Sea & \(10^{4}\) \\ \hline \hline \end{tabular}
\end{table}
Table 1: The table reports the microalgal species cultured for this study, name abbreviation (Abb), their class, growth media, sampling location and cell concentration at the end of the stationary phase when they were pelleted and stored for the successive analyses.  
microalgal extract + 10 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) Pp\(a\), (2) 25 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) microalgal extract + 10 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) Pp\(a\), (3) 25 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) microalgal extract + 5 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) Pp\(a\), (4) 10 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) Pp\(a\). Extracts were dissolved in dimethyl sulfoxide (DMSO; maximum concentration 1% \(\
u/\
u\)) and tested at least in triplicate. After 72 h of incubation, cell viability was evaluated using the MTT assay (3-(4,5-dimethyl-2-thiazolyl))-2,5-diphenyl-2H-tetrazolium bromide; A2231,0001, Applichem Panreac Tischkalender, Darmstadt, GmbH) as in [37]. For JIN3 cells, grown mostly as single cells in suspension, after 72 h of incubation, cell viability was assessed using Orangu\({}^{\text{TM}}\) Cell Counting Solution (OR01-1000) by directly adding it to the medium. After 1 h in the incubator, the absorbance was measured at 450 nm in the same above-mentioned microplate reader. Photo-activation experiments were performed by exposing cells treated with extracts, fractions and/or Pp\(a\) for 1 h under 1.000 Lux\(\cdot\)m\({}^{-2}\), immediately followed by the MTT or Orangu assay.  
The viability of the melanoma cell line A2058 exposed to Pp\(a\) (i.e., 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10 and 50 \(\upmu\)M) and photo-activation for 1 h (to evaluate the combination of Pp\(a\) and light for possible photodynamic therapy) was also evaluated with the MTT protocol. _Cylindotheca clostarium_ (Cc) was the most active species combined with Pp\(a\) and further fractionation and fraction/Pp\(a\) testing was performed (for 24 h). When photo-activation was tested, 1 h light exposure was performed before the MTT assay (as above). Cell survival was expressed as a percentage of viable cells in treated samples, with respect to untreated control cultures with only DMSO.