Opera phenix high content imaging microscope

A549 cells were trypsinized, and 1 × 10³ cells per well were plated in 40 μl fully supplemented medium in 384-well spheroid plates (Corning) using a PerkinElmer JANUS automated liquid handling workstation (PerkinElmer). After 4 d of cultivation, spheroids were stained with 1 μM calcein AM cell-permeant dye and 1 μg/ml Hoechst for 3 h at 37°C. Spheroids were washed with PBS and spun down at 79g for 1 min. A time point zero image was made of the spheroids, PBS was aspirated, therapy was added to each well, and spheroids were briefly spun down at 79g. Wells were imaged directly afterwards every 3 min for 30 consecutive minutes using the PerkinElmer Opera Phenix high-content imaging microscope (PerkinElmer). For each time point, images were taken of each spheroid on five different planes in the z-axis (i.e., 0, 5, 10, 15, and 20 μM). Images were analysed using the image analysis algorithms of Harmony software. The amount of total calcein AM per cell per spheroid was calculated by dividing the amount of calcein AM expression per image by the amount of Hoechst-positive nuclei over all five z-axis planes. The spheroid was subsequently divided over an inner region (30% of spheroid) and an outer region (70% of spheroid) to calculate the out/in ratio and approximate speed of diffusion and toxicity.