Assessing the performance of the Cell Painting assay across different imaging systems

Abstract

Quantitative microscopy is a powerful method for performing phenotypic screens, from which image-based profiling can extract a wealth of information, termed profiles. These profiles can be used to elucidate the changes in cellular phenotypes across cell populations from different patient samples or following genetic or chemical perturbations. One such image-based profiling method is the Cell Painting assay, which provides morphological insight through the imaging of eight cellular compartments. Here, we examine the performance of the Cell Painting assay across multiple high-throughput microscope systems and find that all are compatible with this assay. Furthermore, we determine independently for each microscope system the best performing settings, providing those who wish to adopt this assay an ideal starting point for their own assays. We also explore the impact of microscopy setting changes in the Cell Painting assay and find that few dramatically reduce the quality of a Cell Painting profile, regardless of the microscope used.

JUMP Cell Painting dataset: morphological impact of 136,000 chemical and genetic perturbations

“…The JUMP dataset comprises four subsets: a large production dataset of cells perturbed by three different perturbation modalities–chemical compounds (small molecules), overexpression of genes using Open Reading Frames (ORFs) and gene knockout by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) guides (cpg0016), and three pilot datasets in which cells were perturbed under various experimental and imaging conditions (cpg0000 17 , cpg0001 1 and cpg0002 18 ; note that the first two of these were previously released). We use the term “knockout” here because the majority of resulting alleles have frameshift mutations, preventing further protein production; however, varying levels of proteins may remain at the time of analysis, and some cells will not contain knockout alleles at all.…”

“…In addition, three datasets were generated during the pilot phase of the project (pilot data). These include a previously released compound dataset to test different experimental/staining conditions (cpg0001; CPPilot data 1 ), another previously released dataset to test different perturbation modalities (cpg0000; CPJUMP1 data 17 ) and a compound dataset to test different microscopes (cpg0002; CPJUMP-Scope1 data 18 ). In the following sections, unless specified explicitly, all the details about experimental conditions and analysis pertain to the cpg0016 data.…”

SPACe (Swift Phenotypic Analysis of Cells): an open-source, single cell analysis of Cell Painting data

“…Plates were imaged inside a three-day window on a Yokogawa CV8000 high throughput spinning disk confocal microscope with sequential imaging of the five CP channels and appropriate laser/emission filter combinations as described in (1,31). Imaging was performed with a 20xW/1.0 objective, and a short z-stack (3x z planes, 1µm apart) was captured to correct for plate unevenness.…”

“…We chose a selection of potential reference chemicals (Table 1) that have been previously shown in publications or by the JUMP consortium to alter the phenotype of U2OS cells (3,29,30), including saccharin and sorbitol as negative controls. Cells were treated for 24 hours ahead of the CP protocol, which was performed either manually or robotically following published reports (1)(2)(3) and imaged using a Yokogawa CV8000 high throughput spinning disk confocal using the established conditions (31). Figure 2A shows representative color images of the indicated compounds.…”

Unleashing the potential of cell painting assays for compound activities and hazards prediction

“…Imaging is typically performed using an automated high-content microscope to capture a certain set of image fields. A recent publication by Tromans-Coia et al ( Tromans-Coia et al, 2023 ) investigated the compatibility of the CP assay with various high-content microscopes and summarized specific recommendations for the implementation of the CP assay to a new laboratory. Each well of the 384-well plate is usually imaged at multiple positions, in horizontal (xy) and vertical (z) dimensions, to collect a sufficiently large number of cells and different sub-cellular regions.…”