Arrayscan hcs reader

At 48 h after siRNA treatment, the HUVECs (4×10⁴ cells/well) in the experimental and control groups were resuspended in endothelial complete medium without serum and seeded onto 96-well plates coated with Matrigel (70 μl). Photomicrographs of the center of each well were obtained following incubation of the cells at 37°C for 48 h. The tubes were stained using a Cellomics Cytoskeletal Rearrangement kit and were analyzed with Cellomics ArrayScan (Cellomics, Pittsburgh, PA, USA). Cell images were acquired with the ArrayScan^® HCS Reader (Cellomics, Pittsburgh, PA, USA). Tube formation was assessed by measuring the tube length by using the Image-Pro Plus 6.0 image processing system (Media Cybernetics, Inc., Rockville, MD, USA). Data are expressed as mm/mm².

The effect of AMEAE on the ROS formation in A549 cells was determined by ROS assay. Briefly, treated lung cancer cells with AMEAE at different concentrations in 96-well plates were incubated for 24 h. After the incubation time, the treated cells were stained with dihydroethidium (DEH) at 2.5 μg/mL and Hoechst 33342 (500 nM) dyes for 30 min. Then, cells were fixed with paraformaldehyde (3.5%) for 15 min and washed with PBS twice. The Cellomics ArrayScan HCS reader was used to measure the ROS generation in treated A549 cells.
To further determine the role of ROS generation in AMEAE-induced antiproliferative effect, A549 cells were treated with antioxidants prior to treatment with AMEAE and the cell viability was measured after 24 h. In brief, A549 cells in the exponential phase of growth were supplemented with antioxidants superoxide dismutase (SOD, 300 U/mL) and catalase (400 U/mL) for 1 h prior to AMEAE (20 μg/mL) treatment for 24 h. After incubation time, the cell viability analysis was carried out using a microplate reader (Asys UVM340, Eugendorf, Austria).

The Cellomics Multiparameter Neurite-Outgrowth Kit was used to perform multiple neuronal differentiation assays. According to their protocols, NPCs were plated in a 96-well plate at a concentration of 2 × 10⁴ cells per well, grown in medium in the presence or absence of bFGF (20 ng/ml), and then treated with AG1478 (EGFR tyrosin kinase inhibitor, 10, 20 μM), Cetuximab (EGFR monoclonal antibody, 5, 10 μM), 5-FU (antineoplastic drug, 10, 20 ug/mL), LY294002 (PI3K/AKT inhibitor, 10, 20 μM), and CDP0857 (10, 25 μM) during 24 h. Next, 50 μL of paraformaldehyde 4% was added to each well to fix the cells, and PBS was used to wash the cells. Then, the cells were incubated with 30 μL of primary antibody solution per well for 2 h. The cells were further incubated for 1 h with 30 μL of secondary antibody solution at room temperature. Nuclei were counterstained with DAPI. Cellular screening, data acquisition, and data interpretation were conducted on the ArrayScan HCS Reader (Cellomics Inc., Pittsburgh, PA, USA).