Delfia dna fragmentation assay

Cell viability, apoptosis, and proliferation assays were performed on a single 96-well plate. On the first day, cells were seeded in a complete medium at a concentration of 1.5 × 10⁴ cells per well and incubated for 24 h at 37 °C in 5% CO₂. The complete medium was then changed into 100 µL starvation medium containing 10 µL of 5-bromo-2′-deoxyuridine (BrdU), and the cells were incubated for 24 h at 37 °C in 5% CO₂. Subsequently, 10 µL of PrestoBlue reagent (Thermo Fisher Scientific, Naarden, The Netherlands) was added to the wells, and fluorescence (excitation at 550 nm and emission at 590 nm) was measured eight times at 10 min intervals (control wells: without cells and reagent). Apoptosis and proliferation were detected according to the manufacturer’s protocols using TUNEL assay (DELFIA^® DNA Fragmentation Assay; PerkinElmer, Vaughan, ON, Canada) and BrdU incorporation (DELFIA^® Cell Proliferation Kit; PerkinElmer, Vaughan, ON, Canada), respectively. The VICTOR X4™ Multilabel Plate Reader (PerkinElmer, Vaughan, ON, Canada) was used to measure fluorescence. The assay was performed in triplicate for each cellular variant.

Three cellular assays were performed simultaneously on a single plate: these compared the differences in apoptosis, cell viability and proliferation between the examined variants. Cell line variants were seeded on a 96-well plate at a concentration of 1.5 × 10⁴ cells per well. After 24 h of incubation, medium was changed into 90 µl starving medium with 10 µl 5-bromo-2′-deoxyuridine (BrdU) and the cells were incubated for the next 24 h. Next day, 10 µl of PrestoBlue reagent (Thermo Fisher Scientific, Netherlands) was added to the wells and fluorescence signals (excitation of 550nm and emission of 590 nm) with 10 min intervals were measured. As controls, we also used wells on plate without cells and reagent. Proliferation was detected using BrdU incorporation (DELFIA^® Cell Proliferation Kit; PerkinElmer, Canada) and apoptosis based on TUNEL assay (DELFIA^® DNA Fragmentation Assay; PerkinElmer, Canada). The procedure was conducted according to the manufacturer’s protocol. The fluorescence signal was detected with a VICTOR X4™ Multilabel Plate Reader (PerkinElmer, Canada). The assay was performed in biological triplicate for each cell variant.

A triplex test simultaneously evaluating cellular redox potential (data not shown), proliferation (data not shown), and apoptosis of the cells was used in order to reduce population and systematic discrepancies between cell cultures. Apoptosis was assessed with the DELFIA® DNA fragmentation assay (PerkinElmer), which exploit terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL reaction) and samarium-labeled streptavidin.
Ten thousand cells/well were incubated for 48 h in culture media deprived of phenol red, supplemented with 10% charcoal stripped serum (Gibco) on a white, clear bottom 96-well plates. Cells were then treated either with 10⁻⁸ M 17β-estradiol (Sigma) or vehicle control (i.e., equivalent amounts of ethanol). Samples were collected at time 0 (untreated) and 24 h after treatment.