Juli stage real time cell history recorder

Cell proliferation was measured using CCK8 (Dojindo, Tokyo, Japan), and JuLI™ Stage Real-Time Cell History Recorder (NanoEntek, Seoul, South Korea), the details were described previously 23 (link), 25 (link), 26 . The live cell analyzer monitored cell confluence by recording cell images at 2-hour intervals. On the first day, 2 × 10⁵ cells were seeded in each well. During log growth phase, monitored continuously for 54 h. Cell numbers were counted by CellDrop FL Fluorescence Cell Counter (Devovix, USA).Would healing, and transwell migration analysis were performed as described previously 23 (link), 25 (link). Motility experiments were done according to our previous research 26 . Briefly, cells were harvested and resuspended at a density of 1 × 10⁴ cells/mL in 6-well culture plates, and then images were taken continuously using JuLI™ Stage Real-Time Cell History Recorder (NanoEntek, Seoul, South Korea) for 12 hours with an interval of 15 min. The cell motility ability was quantified via Image-pro.

RFP-expressing endometrial cancer cell spheroids were grown by culturing 100 epithelial cells (Ishikawa RFP⁺ or MFE-296 RFP⁺) in hanging drop fashion [41 (link)] with 3% matrigel. After 72 hr, oncospheres were transferred to 90% confluent monolayer of GFP-expressing endometrial stromal fibroblast cells (HESC GFP⁺). Images were obtained every 24 hr using 10x objective on JuLi^TM Stage Real-Time Cell History Recorder (NanoEnTek) in an incubator at 37°C and humidified 5% CO₂.

The Matrigel ${}^{\mathrm{TM}}$  -embedded cells and spheroids in flat-bottomed or U-bottom 96-well plates were time-lapse-imaged using a JuLI ${}^{\mathrm{TM}}$   stage real-time cell history recorder (NanoEntek, South Korea) placed inside an incubator ( $37{}^{\circ }\text{C}$ and 5% ${\text{CO}}_2$ ). Using the built-in software, exposure time, brightness, and focus were adjusted for each well. Time-lapse bright-field images were obtained using the fully automated x-y-z stage, every 3 min (GA) or 5 min (GFA) for at least 24 h using a 10x objective to obtain a resolution of $440\mathrm{\mu }\text{m/pixel}$ . The recorded images are 2D projections of a 3D migration. If a cell migrates too far away from the focal plane it is not visible anymore. The same goes if the cell migrates outside the field of view^{13 (link),33 (link)}.