Implementación y operación de la Seguridad y Salud en el Trabajo

All the biological studies were performed by Dr. Isolda Romero-Caneón at the University of Warwick. The following sections outline the general cell experiments used in this thesis. More specific experiments will be described in the appropriate chapter.

2.2.15.1. Cell maintenance 20, 21

A2780 human ovarian carcinoma cells were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK). All cells were grown in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% of fetal calf serum, 1 % of 2 mM glutamine and 1 % penicillin/streptomycin and used between passages 5-15. The cells were grown as adherent monolayers at 310 K in a 5 % CO2 humidified atmosphere and passaged at approximately 70-80 % confluence, using 0.25 % trypsin/EDTA.

2.2.15.2. In vitro growth inhibition assay

Stock solutions of the Ru(II) or Rh(III) complexes were freshly prepared in 5 % DMSO and a mixture 0.9 % saline (v/v): medium (1:1 v/v) following serial dilutions in RPMI-1640 to achieve working concentrations. Metal concentrations were determined by ICP-MS.

The antiproliferative activities of the complexes in A2780 ovarian cancer cells were determined. 96-well plates were used to seed 5000 cells per well. The plates were pre-incubated in drug-free media at 310 K for 48 h before addition of various concentrations of the compounds. The drug exposure period was 24 h, after which, the supernatant was removed by suction and each well washed with PBS. A further 48 h was allowed for the cells to recover in drug-free medium at 310 K. The sulforhodamine B (SRB) colorimetric assay was used to determine cell viability. IC50 values, as the concentration which caused 50 % of cell death, were determined as duplicates of triplicates in two independent sets of experiments and their standard deviations were calculated.

The data were analysed using Origin 8.5. IC50 values were obtained from plots of the survival percentage of cells versus the logarithm of the concentration expressed in millimolar units and fitted to a sigmoidal curve. IC50 values of cisplatin were determined in each well- plate as a validation.

Sulforhodamine B assay (SRB) 22

SRB is a pink dye which binds quantitatively to aminoacid residues in acid conditions, however under basic conditions the dye is liberated and can be extracted and analyzed by optical absorption spectroscopy (λ = 568 nm).

To each well, 50 µL of trichloroacetic acid (TCA) (10 % v/v) was added and incubated for an hour at 277 K. The well plates were washed with water 10 times and then dried in air.

Aliquots of 50 µL of SRB (0.4 %, prepared in 1 % acetic acid) were added and the wells left standing for 30 min at ambient temperature. Excess dye was removed by washed with 1 % acetic acid 5 times and the plate left to dry.

A 10 mM of Tris base solution (200 µL, pH 10.5) were added to each well plate and left to stand for 1 h to solubilise the dye. Absorbance was measured with a multi reader at 570 nm.

2.2.15.3. Cell viability modulation experiments

Cell viability assays were carried out in A2780 ovarian cancer cells. These experiments were performed as described above for IC50 determination with the following experimental modifications:

1) Fixed concentration of complexes was 1/3 x IC50

2) Co-administration of the complex with three different concentrations of sodium formate (0.5, 1 and 2 mM). Both solutions (complex and sodium formate) were added to each well independently, but within 5 min of each other

Stock solutions of the Ru(II) or Rh(III) complexes were freshly prepared in 5 % DMSO and a mixture 0.9 % saline : medium (1:1 v/v). The stock solution was further diluted using RPMI-1640 to achieve working concentrations. Metal concentrations were determined by ICP-MS.

Cell viability percentages were determined as duplicates of triplicates in two independent sets of experiments and their standard deviations were calculated.

2.3.

References

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Chapter 3