Cell Painting PLUS imaging data: U2OS biological replicate 3 (imaging cycle 1)

These are supplementary data for Wedler et al. (2026): Cell Painting PLUS: An iterative staining-elution protocol for high-content phenotypic screenings (Current Protocols, doi:10.1002/cpz1.70368). The data represent the first imaging cycle of the third biological replicate in U2OS cells of the Cell Painting PLUS experiment described in von Coburg et al. 2025 (https://doi.org/10.1038/s41467-025-58765-8). Cells were incubated with compounds for 48 h, stained and imaged with five fields per well and two optical sections (z-planes) using Opera Phenix microscope (Revvity Inc.).Channel Hoechst 33342; (Excitation: 405; Emission: 435-480) depicts actin structures ( stained with Alexa Fluor™ Plus 405 Phalloidin dye).Channel Alexa 488 (Excitation: 488, Emission: 500-550) represents RNA (stained with SYTO™ 14 Green Fluorescent Nucleic Acid Stain). Channel Alexa 568 (Excitation: 561; Emission: 570-630) shows mitochondria (stained with MitoTracker Orange CMTMROS). Channel Alexa 647 (Excitation: 640; Emission: 650-760) visualizes lysosomes (stained with Cell Navigator™ Lysosome Staining Kit).The imaging data are presented in the format of a Harmony archive file. The file needs to be imported into the Harmony software (Revvity Inc.) to access the imaging data. The plate layout (including information on compounds and tested concentrations) is described in the Compound_layout.xlsx file. Staining and imaging details are described in in von Coburg et al. 2025 (https://doi.org/10.1038/s41467-025-58765-8).The raw imaging data presented here can be used as input data for further image analysis as described in Wedler et al (2026).本数据集为Wedler等人（2026）的补充数据：《细胞绘画PLUS（Cell Painting PLUS）：用于高内涵表型筛选的迭代染色-洗脱方案》（发表于*Current Protocols*，DOI: 10.1002/cpz1.70368）。本数据对应von Coburg等人2025年发表的细胞绘画PLUS实验中，U2OS细胞第三份生物学重复的第一轮成像循环（原文链接：https://doi.org/10.1038/s41467-025-58765-8）。实验流程如下：细胞经化合物孵育48小时后进行染色与成像，每孔采集5个视野、2个光学切面（z平面），成像设备为Opera Phenix显微镜（Revvity公司出品）。各成像通道信息如下：1. Hoechst 33342（赫斯特33342）通道：激发波长405nm，发射波长435~480nm，用于标记肌动蛋白结构（采用Alexa Fluor™ Plus 405鬼笔环肽染料染色）；2. Alexa 488通道：激发波长488nm，发射波长500~550nm，用于标记RNA（采用SYTO™ 14绿色荧光核酸染料染色）；3. Alexa 568通道：激发波长561nm，发射波长570~630nm，用于标记线粒体（采用MitoTracker Orange CMTMROS染色）；4. Alexa 647通道：激发波长640nm，发射波长650~760nm，用于可视化溶酶体（采用Cell Navigator™溶酶体染色试剂盒染色）。本成像数据以Harmony归档文件格式存储，需导入Harmony软件（Revvity公司出品）方可读取。孔板布局（包含化合物及测试浓度信息）详见Compound_layout.xlsx文件。染色与成像细节详见von Coburg等人2025年的研究（https://doi.org/10.1038/s41467-025-58765-8）。本数据集提供的原始成像数据可作为输入数据，用于执行Wedler等人（2026）中描述的后续图像分析流程。