Sivagurunathan Suganya, Byrne Patrick, Muñoz Alán F, Arevalo John, Carpenter Anne E, Singh Shantanu, Kost-Alimova Maria, Cimini Beth A
Imaging Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
The Center for the Development of Therapeutics, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
SLAS Discov. 2025 Aug 28;36:100268. doi: 10.1016/j.slasd.2025.100268.
Cell Painting, the leading image-based profiling assay, involves staining plated cells with six dyes that mark the different compartments in a cell. Such profiles can then be used to discover connections between samples (whether different cell lines, different genetic treatments, or different compound treatments) as well as to assess particular features impacted by each treatment. Researchers may wish to vary the standard dye panel to assess particular phenotypes, or image cells live while maintaining the ability to cluster profiles overall.
In this study, we evaluate the performance of dyes that can either replace or augment the traditional Cell Painting dyes or enable tracking live cell dynamics. We perturbed U2OS cells with 90 different compounds and subsequently stained them with either standard Cell Painting dyes (Revvity), or with MitoBrilliant (Tocris) replacing MitoTracker or Phenovue phalloidin 400LS (Revvity) replacing phalloidin. We also tested the live-cell compatible ChromaLive dye (Saguaro).
All dye sets effectively separated biological replicates of the same sample vs. negative controls (phenotypic activity), although separating from replicates of all other compounds (phenotypic distinctiveness) proved challenging for all dye sets. While individual dye substitutions within the standard Cell Painting panel had minimal impact on assay performance, the live cell dye exhibited distinct performance profiles across different compound classes compared to the standard panel, with later time points more distinct than earlier ones.
Substituting MitoBrilliant or Phenovue phalloidin 400LS for standard mitochondrial or actin dyes minimally impacted Cell Painting assay performance. Phenovue phalloidin 400LS offers the advantage of isolating actin features from Golgi or plasma membrane while accommodating an additional 568 nm dye. Live cell imaging, enabled by ChromaLive dye, provides real-time assessment of compound-induced morphological changes. Combining this with the standard Cell Painting assay significantly expands the feature space for enhanced cellular profiling. Our findings provide data-driven options for researchers selecting dye sets for image-based profiling.
细胞绘画是领先的基于图像的分析方法，它使用六种染料对培养的细胞进行染色，这些染料可标记细胞内的不同区室。然后，这些图谱可用于发现样本之间的联系（无论是不同的细胞系、不同的基因处理还是不同的化合物处理），以及评估每种处理所影响的特定特征。研究人员可能希望改变标准染料组合来评估特定表型，或者对活细胞进行成像，同时保持整体图谱聚类的能力。
在本研究中，我们评估了能够替代或补充传统细胞绘画染料或实现活细胞动态追踪的染料的性能。我们用90种不同的化合物处理U2OS细胞，随后用标准细胞绘画染料（Revvity）或用MitoBrilliant（Tocris）替代MitoTracker或用Phenovue鬼笔环肽400LS（Revvity）替代鬼笔环肽对细胞进行染色。我们还测试了与活细胞兼容的ChromaLive染料（Saguaro）。
所有染料组合都有效地将同一样本的生物学重复与阴性对照区分开来（表型活性），尽管对所有染料组合来说，将其与所有其他化合物的重复区分开来（表型独特性）具有挑战性。虽然标准细胞绘画组合中的个别染料替代对分析性能影响最小，但与标准组合相比，活细胞染料在不同化合物类别中表现出不同的性能图谱，后期时间点比早期更明显。
用MitoBrilliant或Phenovue鬼笔环肽400LS替代标准的线粒体或肌动蛋白染料对细胞绘画分析性能影响最小。Phenovue鬼笔环肽400LS的优势在于能将肌动蛋白特征与高尔基体或质膜区分开来，同时还能容纳额外的568nm染料。ChromaLive染料实现的活细胞成像可实时评估化合物诱导的形态变化。将其与标准细胞绘画分析相结合可显著扩展特征空间，以增强细胞分析。我们的研究结果为研究人员选择基于图像分析的染料组合提供了数据驱动的选项。
