# The Cytotoxic Activity of Diiron Bis-Cyclopentadienyl Complexes with Bridging C\({}_{3}\)-Ligands
## 4 Experimental
### In Vitro Cytotoxicity Investigation
#### 4.4.2 Cytotoxicity Assay  
Prior to incubation with the selected complex for 72 h, A2780, A2780 cisR, BxPC-3 and A549 cells were seeded in 96 wells tissue culture polystyrene plates at a concentration of 3 \(\times\) 10\({}^{3}\), 6 \(\times\) 10\({}^{3}\), 7 \(\times\) 10\({}^{3}\) and 3 \(\times\) 10\({}^{3}\) cells per well, respectively, and allowed to proliferate for 24 h at 37 \({}^{\circ}\)C in a 5% atmosphere of CO\({}_{2}\). For the assay at 24 h, A2780 and A2780 cisR cells were seeded at a concentration of 1 \(\times\) 10\({}^{4}\) and 1.5 \(\times\) 10\({}^{4}\) cells per well, respectively. Stock solutions of compounds were prepared in DMSO and were sequentially diluted in  
\begin{table}
\begin{tabular}{c|c c c c c c c} \hline \hline \multirow{2}{*}{**Comp.**} & \multicolumn{4}{c}{**DMSO-d\({}_{6}\)/D\({}_{2}\)O**} & \multicolumn{4}{c}{**DMSO-d\({}_{6}\)/DMEM-d**} \\ \cline{2-9}  & **6 h** & **24 h** & **48 h** & **72 h** & **6 h** & **24 h** & **72 h** \\ \hline
**4a** & 3 & 13 & 27 & 70 & 5 & 9 & 100 \\
**4b** & 25 & 71 & 100 & 100 & 13 & 49 & 100 \\
**4c** & 17 & 60 & 88 & 100 & 9 & 20 & 100 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Fraction (%) of diiron complex (measured as released cyclopentadiene, two CpH units being released per molecule [19]) degraded in aqueous solutions stored at 37 \({}^{\circ}\)C after variable times (hours).  
medium (final DMSO concentration of 0.5%) to achieve the final desired concentration (0-100 \(\upmu\)M). Cells incubated with cisplatin (0-100 \(\upmu\)M) were used as positive control. Cells were treated with the different concentration of compounds for 24 or 72 h at 37 \({}^{\circ}\)C in a 5% atmosphere of CO\({}_{2}\). At the end of the incubation time, cell viability was assessed by means of WST-1 tetrazolium salt reagent (Roche). Briefly, cells were incubated for 4 h with WST-1 reagent diluted 1:10, at 37 \({}^{\circ}\)C and 5% CO\({}_{2}\). Measurements of formazan dye absorbance, which directly correlates with the number of viable cells, were carried out with a microplate reader (Biorad, Milan) at 450 nm, using 655 nm as reference wavelength. The 50% inhibitory concentration of tested compound (IC\({}_{50}\)) refers to concentration at which 50% of cell death is observed with respect to the control. All the in vitro biological tests were performed in triplicate. Concentration effect curves were generated by nonlinear regression curves (GraphPad Prism) and the data are reported as mean \(\pm\) standard deviation.  
The following are available online at [[https://www.mdpi.com/article/10.3390/app11104351/s1](https://www.mdpi.com/article/10.3390/app11104351/s1)]([https://www.mdpi.com/article/10.3390/app11104351/s1](https://www.mdpi.com/article/10.3390/app11104351/s1)), Figures S1-S4: NMR spectra of complexes.  
Conceptualization, F.M. and F.C.; methodology, L.B. and S.B.; investigation, L.B., S.B. and G.P. ([PERSON]); data curation, L.B., S.B., F.C. and F.M.; writing--original draft preparation, F.M.; writing--review and editing, L.B., S.B., G.P. ([PERSON]), F.C. and F.M.; supervision, F.C. and F.M.; funding acquisition, L.B., S.B., G.P. ([PERSON]), F.C. and F.M. All authors have read and agreed to the published version of the manuscript.  
This research was funded by University of Pisa, Fondi di Ateneo 2020.  
Not applicable.  
Data Availability Statement: Data is contained within the article or supplementary material.  
The authors declare no conflict of interest.