Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes

Abstract

In morphological profiling, quantitative data are extracted from microscopy images of cells to identify biologically relevant similarities and differences among samples based on these profiles. This protocol describes the design and execution of experiments using Cell Painting, which is a morphological profiling assay that multiplexes six fluorescent dyes, imaged in five channels, to reveal eight broadly relevant cellular components or organelles. Cells are plated in multiwell plates, perturbed with the treatments to be tested, stained, fixed, and imaged on a high-throughput microscope. Next, an automated image analysis software identifies individual cells and measures ∼1,500 morphological features (various measures of size, shape, texture, intensity, and so on) to produce a rich profile that is suitable for the detection of subtle phenotypes. Profiles of cell populations treated with different experimental perturbations can be compared to suit many goals, such as identifying the phenotypic impact of chemical or genetic perturbations, grouping compounds and/or genes into functional pathways, and identifying signatures of disease. Cell culture and image acquisition takes 2 weeks; feature extraction and data analysis take an additional 1-2 weeks.

Figure 1. The Cell Painting assay, in A549 and U2OS cells

The columns display the five channels imaged in the Cell Painting assay protocol (left to right) as imaged using the ImageXpress XLS microscope: Hoechst 33342 (DNA), concanavalin A (ER), SYTO 14 (nucleoli, cytoplasmic RNA), phalloidin (actin) and WGA (Golgi, plasma membrane), and MitoTracker Deep Red (mitochondria). Scale bar is 20 μm. See Table 1 for additional details about the stains and channels imaged.

Figure 2. Overview of the strategy of morphological profiling using an image-based assay

After perturbing, staining, and imaging cells, the open-source software CellProfiler is used to extract 1,000+ morphological features of each cell. The collection of features is known as a profile: it reflects the phenotypic state of the cells in that sample, and can be compared to other profiles to make inferences.

Figure 3. Sample images from a small-molecule Cell Painting experiment using HUVEC cells

Images are shown from a well treated with transfection reagent alone (negative control, top row) and an siRNA-treated well (bottom row), as imaged on an ImageXpress XLS microscope. The first five columns display the five channels imaged in the Cell Painting assay protocol, while the last column illustrates a merged image of the DNA stain (blue) and the nucleolar/cytoplasmic RNA stain (red), with the identified (i.e., segmented) nuclei and cell body outlines resulting from image analysis overlaid in white and magenta, respectively. Scale bar is 20 μm. See Table 1 for details about the stains and channels imaged. Each panel is 6% of a full image; for each well, 9 images are acquired in a 3 × 3 grid, representing ~52% of the well area in total.