Live-Cell Imaging of MCF10A Cells Treated with EGF or PBS

MCF10A cell culture and experimental procedures were conducted based on established methodologies (10.1038/s42003-022-03975-9). For routine maintenance and passaging, cells were cultured in a growth medium composed of DMEM/F12 (Invitrogen, #11330-032) supplemented with 5% horse serum (Sigma, #H1138), 20 ng/ml EGF (R&D Systems, #236-EG), 0.5 µg/ml hydrocortisone (Sigma, #H-4001), 100 ng/ml cholera toxin (Sigma, #C8052), 10 µg/ml insulin (Sigma, #I9278), and 1% Penicillin/Streptomycin (Invitrogen, #15070-063). For experiments involving EGF perturbation, a growth factor-free medium was prepared using DMEM/F12, 5% horse serum, 0.5 µg/ml hydrocortisone, 100 ng/ml cholera toxin, and 1% Pen/Strep.

Cells were cultured to 50–80% confluency before being detached with 0.05% trypsin-EDTA (Thermo Fisher Scientific, #25300-054). Subsequently, 20,000 cells were seeded into 24-well plates (Thermo Fisher Scientific, #267062) coated with collagen-1 (Cultrex, #3442-050-01) in growth medium. After six hours, the cells were rinsed with PBS, and the medium was replaced with growth factor-free medium. Following an 18-hour period of growth factor deprivation, cells were treated with either PBS or 10 ng/ml EGF (R&D Systems, #236-EG).

Phenotypic responses to EGF treatment were assessed through live-cell imaging using the Incucyte S3 microscope (Essen BioScience, #4647), which captured images every 30 minutes over a 24-hour period. The dataset includes an Excel spreadsheet that documents the experimental conditions for each imaged well.