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.MCF10A细胞的培养与实验流程均基于已确立的成熟方法（DOI: 10.1038/s42003-022-03975-9）。 常规培养与传代阶段，细胞采用DMEM/F12培养基（Invitrogen，货号#11330-032）进行培养，该基础培养基添加以下组分：5%胎马血清（Sigma，货号#H1138）、20 ng/ml 表皮生长因子（EGF，R&D Systems，货号#236-EG）、0.5 µg/ml 氢化可的松（Sigma，货号#H-4001）、100 ng/ml 霍乱毒素（Sigma，货号#C8052）、10 µg/ml 胰岛素（Sigma，货号#I9278）以及1% 青霉素-链霉素混合液（Invitrogen，货号#15070-063）。针对涉及EGF扰动的实验，需配制无生长因子培养基，其配方为：DMEM/F12基础培养基、5%胎马血清、0.5 µg/ml 氢化可的松、100 ng/ml 霍乱毒素以及1% 青霉素-链霉素混合液（Pen/Strep）。 实验前将细胞培养至汇合度50%~80%，随后使用0.05% 胰蛋白酶-EDTA（Thermo Fisher Scientific，货号#25300-054）进行细胞消化。取20,000个细胞接种至包被有I型胶原蛋白（Cultrex，货号#3442-050-01）的24孔板（Thermo Fisher Scientific，货号#267062）中，使用前述常规生长培养基培养。培养6小时后，用磷酸盐缓冲液（PBS）冲洗细胞，并更换为无生长因子培养基。经过18小时的生长因子剥夺处理后，分别使用PBS或10 ng/ml EGF（R&D Systems，货号#236-EG）处理细胞。 采用Incucyte S3活细胞成像系统（Essen BioScience，货号#4647）对EGF处理后的细胞表型响应进行评估，该系统每30分钟采集一次图像，总成像时长为24小时。本数据集包含一份Excel表格，用于记录每个成像孔对应的实验条件。