High throughput sampler

The genotoxic MoA of TEMPO and its 3 derivatives was investigated by a method for evaluating multiple endpoints that are associated with DNA damage response pathways (MultiFlow DNA Damage Kit—p53, γ-H2AX, Phospho-Histone H3) according to the manufacturer’s manual (Litron Laboratories, Rochester, New York). Briefly, 4 × 10⁴ TK6 cells were seeded into each well of a 96-well plate and exposed to 14 concentrations of each chemical. After 4- and 24-h of incubation, 25 μl of cells were removed from each well and added to a 96-well U-bottomed plate preloaded with 50 μl of working MultiFlow Kit Labeling solution. Following 30 min incubation at room temperature, flow cytometric analysis was performed using a FACSCanto II flow cytometer equipped with a High Throughput Sampler (BD Biosciences, San Jose, California). Ten thousand total events were acquired or the entire 30 μl volume was exhausted, whichever came first. A total of 5 endpoints were obtained. More specifically, chemical-induced cytotoxicity was calculated using nuclei to counting bead ratios; phosphorylated γ-H2AX and p53 responses were evaluated based on median fluorescence intensity; and the percentages of phosphorylated histone H3 (p-H3) and polyploidy were quantified as the proportion of p-H3-positive and 8n-positive events relative to the total events with 2n and greater DNA content.

Cells were seeded in 96-well plates and washed twice with sterile PBS after the 24-h treatment. Ethidium monoazide and SYTOX® Green were used to label apoptotic/necrotic cells and chromatin, respectively, following the protocol described in the In Vitro MicroFlowTM Kit (Litron Laboratories, Rochester, NY). After cell lysis, the high-throughput (HT) micronucleus analysis was performed using a FACSCanto II flow cytometer equipped with a High Throughput Sampler (BD Biosciences, San Jose, CA). The stopping gate was set to record 10,000 intact nuclei and threshold parameters were set as recommended by the manufacturer. The percentage of micronuclei (%MN) was calculated as the ratio of micronucleus events to the total number of nucleated events. The data were expressed as the fold-change of %MN in treated groups over vehicle controls. Cell cycle data were collected by retrieving histograms of SYTOX® Green nucleic acid stain fluorescence. The analysis was conducted using FlowJo® (v10.1) with Watson pragmatic model.

Cells were grown from saturation for 5 h in exponential phase SDC at 30 °C. 200 μL of culture (OD₆₀₀ 0.05) were transferred to a shallow 96-well plate (Corning). Estradiol and progesterone were added at the concentrations indicated in Figure 4 and cells were left shaking at 1200 rpm and 30 °C (Thermo-shaker DTS-4, ELMI). After 5 h, cultures were diluted 1:2 with 1× TE buffer and taken to a cytometer (LSR-II, BD Biosciences) equipped with a high throughput sampler (BD Biosciences). Data was collected in FACS DIVA and analyzed in Matlab. Cell density was estimated from flow cytometry event rate.
Light microscopy was conducted on samples uninduced and fully induced with both hormones. 100 μL of cultures treated for 5 h were sonicated and added to glass-bottom plate precoated with conavalin-A. Cells were immediately imaged in a Nilon Eclipse-Ti microscope with a 40× objective.