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

Updates to the protocol

See the wiki for an up-to-date version of the Cell Painting protocol.

Abstract

In morphological profiling, quantitative data are extracted from microscopy images of cells; the goal is 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, a morphological profiling assay multiplexing six fluorescent dyes imaged in five channels, to reveal eight broadly relevant cellular components or organelles. Automated image analysis software identifies individual cells and measures over 1000 morphological features (various measures of size, shape, texture, intensity, etc.) to produce a rich profile suitable for detecting 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.

Data files

1. Raw image data from a RNAi Cell Painting knockdown experiment applied to U2OS cells (doi). [LINK]

2. A sample ImageXpress plate acquisition settings file. [ZIP, 26 KB]

3. CellProfiler pipelines described in the protocol: Illumination correction, quality control, and feature extraction. Please see the module notes for Cell Painting-specific documentation. Pipelines were created using CellProfiler 2.1.1. [ZIP, 50 KB]

4. Example illumination correction images generated by the illumination correction pipeline in (3) for the images provided in (1). [ZIP, 7.7 MB]

5. Listing of per-cell image features generated by CellProfiler using the analysis pipeline in (3). [PDF].

6. Files containing an example morphological profiling dataset generated with the image data provided in (1) and the pipelines supplied in (3).

• Configuration file generated by CellProfiler. [gzip, 26 KB]

• Table of per-cell morphological features. [gzip SQL dump file, 5.4 GB, MD5 checksum: 5ad65c115c699c289854989aa0c6ceab]

• Table of per-cytoplasm morphological features. [gzip SQL dump file, 5.3 GB, MD5 checksum: a98955c94bff5977912b63c568023ac6]

• Table of pable of per-nuclei morphological features. [gzip SQL dump file, 5.5 GB, MD5 checksum: 1c8a2d13a28c16fe86b7fa18cf1fb4a9]

• Per-image table for indexing per-object features. [gzip SQL dump file, 3.4 GB, MD5 checksum: 83073ca85c099558a3c0cac9d06f054f]

• Per-object view of cell, cytoplasm, and nuclei tables. [gzip SQL dump file, 74 KB]

7. Steps to produce per-well morphological profiles from single cell measurements, from the raw image data provided in (1). Sample output is provided in (6) for comparision. [PDF]

8. CSV of per-well profiles generated by running the steps described in (7) on the profiling dataset in (6). [ZIP, 41 MB]