HeLa‐S3 and Hep‐G2
Human cervix carcinoma cell line HeLa‐S3 and human liver carcinoma cell line Hep‐G2 were obtained from European Collection of Cell Cultures (ECACC) and were cultured as described previously.184
HeLa‐S3 and Hep‐G2 cells were cultivated at 37°C in humidified 5% CO2 atmosphere using Dulbecco’s DMEMmedia (Invitrogen) containing 10% fetal calf serum, 1% penicillin and 1%
streptomycin. Cells were split every three days. Both cell lines were tested for mycoplasma infections using a mycoplasma detection kit (Roche Applied Science).
Caco‐2
The human colon cancer cell line Caco‐2 (ATCC HTB‐37) was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and was cultured as described previously.245 Cells were grown in Iscove’s Modified Dulbecco’s Media supplied with 10% fetal calf serum, 50 mg mL‐1 gentamicin at 37°C and 5% CO2. Cells were split every three days.
9.2) AllamarBlue assay
To measure the cell viability we adapted the previously described AlamarBlue assay184,185 AlamarBlue (BioSource Europe), the dark blue coloured sodium salt of resazurin (7‐hydroxy‐
3H‐phenoxazin‐3‐one‐10‐oxide) was used to measure growth and viability of cells which are capable of reducing it to the fluorescent, pink colored resorufin (7‐hydroxy‐3H‐phenoxazin‐
3‐one). Cells were seeded in 96‐well plates (4,000 HeLa‐S3 cells per well or 8,000 Hep‐G2 cells per well) and allowed to attach for 24 h. Small‐molecules to be tested were dissolved in a suitable amount of DMSO. Different concentrations were prepared by serial dilution with medium to give final concentrations with a maximum DMSO content of 1%. The cells were then incubated for 48 h with 100 μL each of above dilution series. Alamar Blue (10 μL) was added and the cells were incubated for another hour. After excitation at 530 nm, fluorescence at 590 nm was measured using a FL600 Fluorescence Microplate Reader
(Bio‐TEK). Cell viability is expressed in percent with respect to a control containing only pure medium and 1% DMSO incubated under identical conditions. All experiments were repeated for a minimum of three times with each experiment done in four replicates. The resulting curves were fitted using the software Prism 4 (GraphPad).
9.2) MTT assay
To measure the cell viability we adapted the previously described 3‐(4,5‐dimethylthiazol‐2‐
yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay.188,189 2,000 cells per well were seeded in a 96‐well plate format and allowed to recover overnight. On the next day, different concentrations of small‐molecules to be tested or DMSO controls were prepared by serial dilution with medium to give final concentrations with a maximum DMSO content of 1%. The dilutions of the compounds were added and the cells were pre‐incubated for 24 h. The following day, different concentrations of DNA damaging agents were tested (H2O2, TMZ) or controls (DMSO for TMZ) were prepared by serial dilution with medium to give final concentrations with a maximum DMSO content of 1% (DMSO of small‐molecules dilution series included). Different dilutions of the agents were added and the cells were incubated for additional 72 h. Afterwards the medium was replaced with fresh culture medium containing 0.5 mg mL‐1 MTT. The cells were incubated another 2.5 h. Then, the MTT medium was discarded again and replaced by DMSO to dissolve the formazan. Absorbance was measured at 565 nm using a microplate reader (TECAN Sunrise Remote). Cell viability (or cell death respectively) was expressed as a percentage of according controls. The resulting diagrams and curves were generated using the software Prism 4 (GraphPad).
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