Arrayscan vti hcs reader

MCF-7 and BT20 cells were seeded in a 96-well plate (Corning, USA) at a density of 8,000 cells/well and then treated with various concentrations (10, 32, 100 μM) of DMDD for 2 h, followed by incubation with 10 ng/ml tumor necrosis factor-alpha (TNF-α) (Sigma-Aldrich, USA) for 30 min. Untreated cells and cells treated with 10 ng/ml TNF-α alone served as controls. Cells were fixed, permeabilized, and sequentially stained with NF-κB p65 primary antibody (Cell Signaling Technology, USA), DyLight 488-conjugated secondary antibody, and Hoechst 33342 dye. The Hoechst and DyLight fluorophores detect changes in nuclear morphology (blue fluorescence) and NF-κB distribution (green fluorescence), respectively. The samples were analyzed on an Arrayscan VTI HCS Reader (Thermo Scientific, USA). The Nuclear Translocation BioApplication (Thermo Scientific, USA) was used for image acquisition and analysis. For each well, at least 400 cells were automatically acquired and analyzed. The translocation index was calculated by measuring the average intensity difference of NF-κB between the identified cytoplasmic region and nuclear region (MEAN_CircRingAvgIntenDiffCh2).

The fixed cells were washed with PBS and permeabilized with 0.25% Triton X-100 in PBS for 8 min. The primary and secondary antibodies were diluted in PBS containing 5% bovine serum albumin. The antibodies were as follows: 0.2 µg/mL of goat anti-Oct6 and 2 µg/mL of Alexa Fluor 594 rabbit anti-goat IgG. To measure the intensity of Oct6 expression, wide-field fluorescence images from four fields in each well of the 384-well plate were acquired with the ArrayScan VTI HCS Reader (Thermo Fisher Scientific, Waltham, MA, USA) using a 10× objective lens. The images were analyzed with the target activation V3 BioApplication (Thermo Fisher Scientific). 4′,6′-Diamidino-2-phenylindole was used to stain the nucleus.