Computational protocol: Cytotoxic Properties of Titanocenyl Amides on Breast Cancer Cell Line MCF-7

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Protocol publication

[…] Biological activity was determined using the MTT assay originally described by Mossman [] but using 10% Triton in isopropanol as a solvent for the MTT formazan crystals []. HT29 and MCF7 cells were maintained at 37°C and 95% Air/5% CO2 in McCoy's 5A (ATCC) complete medium, which had been supplemented with 10% (v/v) fetal bovine serum (ATCC) and 1% (v/v) antibiotic/antimycotic (Sigma). Asynchronously growing cells were seeded at 1.5 × 104 cells per well in 96-well plates containing 100 μL of complete growth medium, and allowed to recover overnight. Various concentrations of the complexes (1–1300 μM) dissolved in 5% DMSO/95% Medium were added to the wells (eight wells per concentration; experiments performed in quadruplicate plates). The complexes' solutions were prepared first by dissolving the corresponding titanocenyl in DMSO and then Medium was added to a final composition of 5% DMSO/95% Medium. In addition to the cells treated with the titanocenyls, two controls experiments were run: one without any addition of solvent mixture (5% DMSO/95% Medium) and one adding 5% DMSO/95% Medium to the cells. Both control experiments behaved identical, showing that 5% of DMSO in the Medium did not have any toxic effect on the cell growth. The cells were incubated for an additional 70 hours. After this time, MTT dissolved in complete growth medium was added to each well to a final concentration of 1.0 mg/mL and incubated for two additional hours. After this period of time, all MTT containing medium was removed; cells were washed with cold PBS and dissolved with 200 μL of a 10% (v/v) Triton X-100 solution in isopropanol. After complete dissolution of the formazan crystals, well absorbances were recorded in triplicates on a 340 ATTC Microplate Reader (SLT Lab Instruments) at 570 nm with background subtraction at 630 nm. Concentrations of compounds required to inhibit cell proliferation by 50% (IC50) were calculated by fitting data to a four-parameter logistic plot by means of SigmaPlot software from SPSS. […]

Pipeline specifications

Software tools SigmaPlot, SPSS
Application Miscellaneous
Organisms Chrysanthemum x morifolium
Diseases Breast Neoplasms
Chemicals Amides